Oligomeric compounds and compositions for use in modulation of small non-coding RNAS

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression and function of small non-coding RNAs. The compositions comprise oligomeric compounds, targeted to small non-coding RNAs. Methods of using these compounds for modulation of small non-coding RNAs as well as downstream targets of these RNAs and for diagnosis and treatment of disease associated with small non-coding RNAs are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 10/909,125 filed Jul. 30, 2004, which claims priority to U.S. provisional application Ser. No. 60/492,056 filed Jul. 31, 2003, Ser. No. 60/516,303 filed Oct. 31, 2003, Ser. No. 60/531,596 filed Dec. 19, 2003, and Ser. No. 60/562,417 filed Apr. 14, 2004, each which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulation of small non-coding RNAs. In particular, this invention relates to compounds, particularly oligomeric compounds, which, in some embodiments, hybridize with or sterically interfere with nucleic acid molecules comprising or encoding small non-coding RNA targets. Such compounds are shown herein to modulate the levels of small non-coding RNAs. The oligomeric compounds of the invention may include one or more modifications thereon resulting in differences in physical or chemical properties compared to unmodified nucleic acids. These modified oligomeric compounds are used as single compounds or in compositions to modulate or mimic the targeted nucleic acid comprising or encoding the small non-coding RNA. In some embodiments of the invention, modifications include chemical modifications that improve activity of the oligomeric compound. In some embodiments, the modifications include moieties that modify or enhance the pharmacokinetic or pharmacodynamic properties, stability or nuclease resistance of the oligomeric compound. In some embodiments, the modifications render the oligomeric compounds capable of sterically interfering with the natural processing of the nucleic acids comprising or encoding the small non-coding RNA targets.

BACKGROUND OF THE INVENTION

RNA genes were once considered relics of a primordial “RNA world” that was largely replaced by more efficient proteins. More recently, however, it has become clear that non-coding RNA genes produce functional RNA molecules with important roles in regulation of gene expression, developmental timing, viral surveillance, and immunity. Not only the classic transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), but also small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), small interfering RNAs (siRNAs), tiny non-coding RNAs (tncRNAs), repeat-associated small interfering RNAs (rasiRNAs) and microRNAs (miRNAs) are now believed to act in diverse cellular processes such as chromosome maintenance, gene imprinting, pre-mRNA splicing, guiding RNA modifications, transcriptional regulation, and the control of mRNA translation (Eddy, Nat. Rev. Genet., 2001, 2, 919-929; Kawasaki and Taira, Nature, 2003, 423, 838-842; Aravin, et al., Dev. Cell, 2003, 5, 337-350). RNA-mediated processes are now also believed to direct heterochromatin formation, genome rearrangements, and DNA elimination (Cerutti, Trends Genet., 2003, 19, 39-46; Couzin, Science, 2002, 298, 2296-2297).

The recently described phenomenon known as RNA interference (RNAi) is involves the processing of double stranded RNA into siRNAs by an RNase III-like dsRNA-specific enzyme known as Dicer (also known as helicase-moi) which are then incorporated into a ribonucleoprotein complex, the RNA-induced silencing complex (RISC). RISC is believed to use the siRNA molecules as a guide to identify complementary RNAs, and an endoribonuclease (to date unidentified) cleaves these target RNAs, resulting in their degradation (Cerutti, Trends Genet., 2003, 19, 39-46; Grishok et al., Cell, 2001, 106, 23-34). In addition to the siRNAs, a large class of small non-coding RNAs known as microRNAs (miRNAs, originally termed stRNA for “short temporal RNAs”) is believed to play a role in regulation of gene expression employing some of the same players involved in the RNAi pathway (Novina and Sharp, Nature, 2004, 430, 161-164).

Like siRNAs, miRNAs are believed to be processed endogenously by the Dicer enzyme, and are approximately the same length, and possess the characteristic 5′-phosphate and 3′-hydroxyl termini. The miRNAs are also incorporated into a ribonucleoprotein complex, the miRNP, which is similar, and may be identical to the RISC (Bartel and Bartel, Plant Physiol., 2003, 132, 709-717). More than 200 different miRNAs have been identified in plants and animals (Ambros et al., Curr. Biol., 2003, 13, 807-818).

In spite of their biochemical and mechanistic similarities, there are also some differences between siRNAs and miRNAs, based on unique aspects of their biogenesis. siRNAs are generated from the cleavage of long exogenous or possibly endogenous dsRNA molecules, such as very long hairpins or bimolecular duplexed dsRNA, and numerous siRNAs accumulate from both strands of dsRNA precursors. In contrast, mature miRNAs appear to originate from long endogenous primary miRNA transcripts (also known as pri-miRNAs, pri-mirs or pri-pre-miRNAs) that are often hundreds of nucleotides in length (Lee, et al., EMBO J., 2002, 21(17), 4663-4670).

The current model of miRNA processing involves primary miRNA transcripts being processed by a nuclear enzyme in the RNase III family known as Drosha, into approximately 70 nucleotide-long pre-miRNAs (also known as stem-loop structures, hairpins, pre-mirs or foldback miRNA precursors) which are subsequently processed by the Dicer RNase into mature miRNAs, approximately 21-25 nucleotides in length. It is believed that, in processing the pri-miRNA into the pre-miRNA, the Drosha enzyme cuts the pri-miRNA at the base of the mature miRNA, leaving a 2-nt 3′ overhang (Ambros et al., RNA, 2003, 9, 277-279; Bartel and Bartel, Plant Physiol., 2003, 132, 709-717; Shi, Trends Genet., 2003, 19, 9-12; Lee, et al., EMBO J., 2002, 21(17), 4663-4670; Lee, et al., Nature, 2003, 425, 415-419). The 3′ two-nucleotide overhang structure, a signature of RNaseIII cleavage, has been identified as a critical specificity determinant in targeting and maintaining small RNAs in the RNA interference pathway (Murchison, et al., Curr. Opin. Cell Biol., 2004, 16, 223-9). Both the primary RNA transcripts (pri-miRNAs) and foldback miRNA precursors (pre-miRNAs) are believed to be single-stranded RNA molecules with at least partial double-stranded character, often containing smaller, local internal hairpin structures. Primary miRNA transcripts may be processed such that one single-stranded mature miRNA molecule is generated from one arm of the hairpin-like structure of the pri-miRNA. Alternatively, a polycistronic pri-miRNA may contain multiple pre-miRNAs, each processed into a different, single-stranded mature miRNA.

Naturally occurring miRNAs are characterized by imperfect complementarity to their target sequences. Artificially modified miRNAs with sequences completely complementary to their target RNAs have been designed and found to function as double stranded siRNAs that inhibit gene expression by reducing RNA transcript levels. Synthetic hairpin RNAs that mimic siRNAs and miRNA precursor molecules were demonstrated to target genes for silencing by degradation and not translational repression (McManus et al., RNA, 2002, 8, 842-850).

Tiny non-coding RNA (tncRNA), one class of small non-coding RNAs (Ambros et al., Curr. Biol., 2003, 13, 807-818) produce transcripts similar in length (20-21 nucleotides) to miRNAs, and are also thought to be developmentally regulated but, unlike miRNAs, tncRNAs are reportedly not processed from short hairpin precursors and are not phylogenetically conserved. Although none of these tncRNAs are reported to originate from miRNA hairpin precursors, some are predicted to form potential foldback structures reminiscent of pre-miRNAs; these putative tncRNA precursor structures deviate significantly from those of pre-miRNAs in key characteristics, i.e., they exhibit excessive numbers of bulged nucleotides in the stem or have fewer than 16 base pairs involving the small RNA (Ambros et al., Curr. Biol., 2003, 13, 807-818).

Recently, another class of small non-coding RNAs, the repeat-associated small interfering RNAs (rasiRNAs) has been isolated from Drosophila melanogaster. The rasiRNAs are associated with repeated sequences, transposable elements, satellite and microsatellite DNA, and Suppressor of Stellate repeats, suggesting that small RNAs may participate in defining chromatin structure (Aravin, et al., Dev. Cell, 2003, 5, 337-350).

A total of 201 different expressed RNA sequences potentially encoding novel small non-messenger species (smRNAs) has been identified from mouse brain cDNA libraries. Based on sequence and structural motifs, several of these have been assigned to the snoRNA class of nucleolar localized molecules known to act as guide RNAs for rRNA modification, whereas others are predicted to direct modification within the U2, U4, or U6 small nuclear RNAs (snRNAs). Some of these newly identified smRNAs remained unclassified and have no identified RNA targets. It was suggested that some of these RNA species may have novel functions previously unknown for snoRNAs, namely the regulation of gene expression by binding to and/or modifying mRNAs or their precursors via their antisense elements (Huttenhofer et al., Embo J., 2001, 20, 2943-2953).

To date, the binding and regulatory sites within nucleic acid targets of the small non-coding RNAs are largely unknown, although a few putative motifs have been suggested to exist in the 3′UTR of certain genes (Lai and Posakony, Development, 1997, 124, 4847-4856; Lai, et al., Development, 2000, 127, 291-306; Lai, Nat. Genet. 2002, 30(4), 363-364).

One miRNA is also believed to act as a cell death regulator, implicating it in mechanisms of human disease such as cancer. Recently, the Drosophila mir-14 miRNA was identified as a suppressor of apoptotic cell death and is required for normal fat metabolism. (Xu et al., Curr. Biol., 2003, 13, 790-795).

Downregulation or deletion of other miRNAs has been associated with B-cell chronic lymphocytic leukemia (B-CLL) (Calin et al., Proc. Natl. Acad. Sci. USA, 2002, 99, 15524-15529), and human homologues of the murine mir-143 and mir-145 mature miRNAs were recently reported to be expressed and processed at reduced steady-state levels at the adenomatous and cancerous stages of colorectal neoplasia (Michael, et al., Mol. Cancer. Res., 2003, 1, 882-891).

Expression of the human mir-30 miRNA specifically blocked the translation in human cells of an mRNA containing artificial mir-30 target sites. In these studies, putative miRNAs were excised from transcripts encompassing artificial miRNA precursors and could inhibit the expression of mRNAs containing a complementary target site. These data indicate that novel miRNAs can be readily produced in vivo and can be designed to specifically inactivate the expression of selected target genes in human cells (Zeng et al., Mol. Cell, 2002, 9, 1327-1333).

Disclosed and claimed in PCT Publication WO 03/029459 are miRNAs from several species, or a precursor thereof, a nucleotide sequence which is the complement of said nucleotide sequence which has an identity of at least 80% to said sequence; and a nucleotide sequence which hybridizes under stringent conditions to said sequence. Also claimed is a pharmaceutical composition containing as an active agent at least one of said nucleic acid and optionally a pharmaceutically acceptable carrier, and a method of identifying microRNA molecules or precursor molecules thereof comprising ligating 5′- and 3′-adapter molecules to the ends of a size-fractionated RNA population, reverse transcribing said adapter containing RNA population and characterizing the reverse transcription products (Tuschl et al., Genes Dev., 1999, 13, 3191-3197).

Small non-coding RNA-mediated regulation of gene expression is an attractive approach to the treatment of diseases as well as infection by pathogens such as bacteria, viruses and prions and other disorders associated with RNA expression or processing.

Consequently, there remains a long-felt need for agents that regulate gene expression via the mechanisms mediated by small non-coding RNAs. Identification of modified miRNAs or miRNA mimics that can increase or decrease gene expression or activity is therefore desirable.

The present invention therefore provides oligomeric compounds and methods useful for modulating gene levels, expression, function or pathways, including those relying on mechanisms of action such as RNA interference and dsRNA enzymes, as well as antisense and non-antisense mechanisms. One having skill in the art, once armed with this disclosure will be able, without undue experimentation, to identify compounds, compositions and methods for these uses.

SUMMARY OF THE INVENTION

The present invention provides oligomeric compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to or mimic nucleic acids comprising or encoding small non-coding RNAs, and which act to modulate the levels of small non-coding RNAs, or interfere with their function.

The present invention also provides oligomeric compounds comprising a first strand and a second strand wherein at least one strand contains a modification and wherein a portion of one of the oligomeric compound strands is capable of hybridizing to a small non-coding RNA target nucleic acid.

The present invention also provides oligomeric compounds comprising a first region and a second region and optionally a third region wherein at least one region contains a modification and wherein a portion of the oligomeric compound is capable of hybridizing to a small non-coding RNA target nucleic acid.

The present invention also provides oligomeric compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding human Dicer, and which act to modulate the levels of the human Dicer RNase III enzyme and interfere with its function, as well as modulating the levels of small non-coding RNAs.

Pharmaceutical and other compositions comprising the compounds of the invention are also provided.

Also provided are methods of screening for modulators of small non-coding RNAs and methods of modulating the levels of small non-coding RNAs in cells, tissues or animals comprising contacting said cells, tissues or animals with one or more of the compounds or compositions of the invention.

Methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of small non-coding RNAs are also set forth herein. Such methods comprise optionally identifying such an animal, and administering a therapeutically or prophylactically effective amount of one or more of the compounds or compositions of the invention to the animal or person.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the interaction of the mir-143 miRNA with three novel binding sites in the ERK5 mRNA coding sequence (GenBank Accession NM_(—)139032.1) identified herein, along with their bimolecular hybridization free energies.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides oligomeric compounds useful in, for example, the modulation of expression, endogenous levels or the function of small non-coding RNAs. As used herein, the term “small non-coding RNA” is used to encompass, without limitation, a polynucleotide molecule ranging from about 17 to about 450 nucleotides in length, which can be endogenously transcribed or produced exogenously (chemically or synthetically), but is not translated into a protein. Small non-coding RNAs may include isolated single-, double-, or multiple-stranded molecules, any of which may include regions of intrastrand nucleobase complementarity, said regions capable of folding and forming a molecule with fully or partially double-stranded or multiple-stranded character based on regions of perfect or imperfect complementarity. Examples of small non-coding RNAs include, but are not limited to, primary miRNA transcripts (also known as pri-pre-miRNAs, pri-mirs and pri-miRNAs, which range from around 70 nucleotides to about 450 nucleotides in length and often taking the form of a hairpin structure); pre-miRNAs (also known as pre-mirs and foldback miRNA precursors, which range from around 50 nucleotides to around 110 nucleotides in length); miRNAs (also known as microRNAs, Mirs, miR5, mirs, and mature miRNAs, and generally refer either to double-stranded intermediate molecules around 17 to about 25 nucleotides in length, or to single-stranded miRNAs, which may comprise a bulged structure upon hybridization with a partially complementary target nucleic acid molecule); or mimics of pri-miRNAs, pre-miRNAs or miRNAs. Small non-coding RNAs can be endogenously transcribed in cells, or can be synthetic oligonucleotides, in vitro transcribed polynucleotides or nucleic acid oligomeric compounds expressed from vectors. Pri-miRNAs and pre-miRNAs, or mimics thereof, may be processed into smaller molecules.

As used herein, the term “miRNA precursor” is used to encompass, without limitation, primary RNA transcripts, pri-miRNAs and pre-miRNAs.

In some embodiments, pri-miRNAs, or mimics thereof, are 70 to 450 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449 or 450 nucleobases in length, or any range therewithin.

In some embodiments, pri-miRNAs, or mimics thereof, are 110 to 430 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429 or 430 nucleobases in length, or any range therewithin.

In some embodiments, pri-miRNAs, or mimics thereof, are 110 to 280 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279 or 280 nucleobases in length, or any range therewithin.

In some embodiments, pre-miRNAs, or mimics thereof, are 50 to 110 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 70, 71 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 or 110 nucleobases in length, or any range therewithin. In some embodiments, pre-miRNAs, or mimics thereof, are 60 to 80 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length, or any range therewithin.

In some embodiments, miRNAs, or mimics thereof, are 15 to 49 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 nucleobases in length, or any range therewithin. In some embodiments, miRNAs, or mimics thereof, are 17 to 25 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleobases in length, or any range therewithin.

Oligomeric compounds of the invention modulate the levels, expression or function of small non-coding RNAs by hybridizing to a nucleic acid comprising or encoding a small non-coding RNA nucleic acid target resulting in alteration of normal function by, for example, facilitating destruction of the small non-coding RNA through cleavage, by sequestration, or by sterically occluding the function of the small non-coding RNA. Further, modified synthetic oligomeric compounds of the present invention may be designed to mimic endogenous small non-coding RNAs. These modifications include, but are not limited to, improved pharmacokinetic or pharmacodynamic properties, binding affinity, stability, charge, localization or uptake. Synthetic mimics can therefore act as replacements for small non-coding RNAs, as competitive inhibitors of naturally occurring small non-coding RNAs or as delivery systems wherein the mimic construct contains one or more functional components.

As used herein, the terms “target nucleic acid,” “target RNA,” “target RNA transcript” or “nucleic acid target” are used to encompass any nucleic acid capable of being targeted including, without limitation, RNA (including microRNAs, stRNAs, small nuclear RNAs, small nucleolar RNAs, small ribosomal RNAs, small hairpin RNAs, endogenous antisense RNAs, guide RNAs, tiny noncoding RNAs, small single or double stranded RNAs that are encoded by heterochromatic repeats at centromeres or other chromosomal origin, and any precursors thereof). These nucleic acid targets can be coding or non-coding sequences; pre-mRNAs or mRNAs; single- or double-stranded, or single-stranded with partial double-stranded character; may occur naturally within introns or exons of messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), or transfer RNAs (tRNAs); and can be endogenously transcribed or exogenously produced.

In some embodiments of this invention, modulation of small non-coding RNA levels, expression or function is achieved via oligomeric compounds which target a further RNA associated with the particular small non-coding RNA. This association can be a physical association between that RNA and the particular small non-coding RNA such as, but not limited to, in an RNA or ribonucleoprotein complex. This association can also be within the context of a biological pathway, such as but not limited to, the regulation of levels, expression or function of a protein-encoding mRNA or its precursor by a small non-coding RNA. As such, the invention provides for modulation of the levels, expression or function of a target nucleic acid where the target nucleic acid is a messenger RNA whose expression levels and/or function are associated with one or more small non-coding RNAs. The messenger RNA function or processing may be disrupted by degradation through an antisense mechanism, including but not limited to, RNA interference, or RNase H, as well as other mechanisms wherein double stranded nucleic acid structures are recognized and degraded, cleaved, sterically occluded, sequestered or otherwise rendered inoperable.

The compounds or compositions of the present invention may also interfere with the function of endogenous RNA molecules. The functions of RNA to be interfered with can include, for example, nuclear events such as replication or transcription as the compounds of the present invention could target or mimic small non-coding RNAs in these cellular processes. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be interfered with can include cytoplasmic events such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, RNA signaling and regulatory activities, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA as the compounds of the present invention could target or mimic small non-coding RNAs in these cellular processes.

In the context of the present invention, “modulation” and “modulation of expression” mean either an increase (stimulation) or a decrease (inhibition) in the amount or levels of a small non-coding RNA, nucleic acid target, an RNA or protein associated with a small non-coding RNA, or a downstream target of the small non-coding RNA (e.g., a mRNA representing a protein-coding nucleic acid that is regulated by a small non-coding RNA). Inhibition is a suitable form of modulation and small non-coding RNA is a suitable target nucleic acid.

In the context of the present invention, “modulation of function” means an alteration in the function of the small non-coding RNA or an alteration in the function of any cellular component with which the small non-coding RNA has an association or downstream effect.

The present invention provides, inter alia, oligomeric compounds and compositions containing the same wherein the oligomeric compound includes one or more modifications that render the compound capable of supporting modulation of the levels, expression or function of the small non-coding RNA by a degradation or cleavage mechanism.

The present invention also provides methods of maintaining a pluripotent stem cell comprising contacting the cell with an effective amount of an oligomeric compound targeting human Dicer. The pluripotent stem cell can be present in a sample of cord blood or bone marrow, or may be present as part of a cell line. In addition, the pluripotent stem cell can be an embryonic stem cell.

The present invention also provides oligomeric compounds and compositions containing the same wherein the oligomeric compound includes one or more modifications that render the compound capable of blocking or interfering with the levels, expression or function of one or more small non-coding RNAs by steric occlusion.

The present invention also provides oligomeric compounds and compositions containing the same wherein the oligomeric compound includes one or more modifications or structural elements or motifs that render the compound capable of mimicking or replacing one or more small non-coding RNAs.

Oligomeric Compounds

In the context of the present invention, the term “oligomeric compound(s)” refers to polymeric structures which are capable of hybridizing to at least a region of a small non-coding RNA molecule or a target of small non-coding RNAs, or polymeric structures which are capable of mimicking small non-coding RNAs. The term “oligomeric compound” includes, but is not limited to, compounds comprising oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics and combinations of these. Oligomeric compounds also include, but are not limited to, antisense oligomeric compounds, antisense oligonucleotides, siRNAs, alternate splicers, primers, probes and other compounds that hybridize to at least a portion of the target nucleic acid. Oligomeric compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular and may also include branching. Separate oligomeric compounds can hybridize to form double stranded compounds that can be blunt-ended or may include overhangs on one or both termini. In general, an oligomeric compound comprises a backbone of linked monomeric subunits where each linked monomeric subunit is directly or indirectly attached to a heterocyclic base moiety. The linkages joining the monomeric subunits, the sugar moieties or sugar surrogates and the heterocyclic base moieties can be independently modified giving rise to a plurality of motifs for the resulting oligomeric compounds including hemimers, gapmers and chimeras.

As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base moiety. The two most common classes of such heterocyclic bases are purines and pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. The respective ends of this linear polymeric structure can be joined to form a circular structure by hybridization or by formation of a covalent bond. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded structure. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide. The normal internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

In the context of this invention, the term “oligonucleotide” refers generally to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside linkages. The term “oligonucleotide analog” refers to oligonucleotides that have one or more non-naturally occurring portions which function in a similar manner to oligonucleotides. Such non-naturally occurring oligonucleotides are often selected over naturally occurring forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for other oligonucleotides or nucleic acid targets and increased stability in the presence of nucleases.

In the context of this invention, the term “oligonucleoside” refers to nucleosides that are joined by internucleoside linkages that do not have phosphorus atoms. Internucleoside linkages of this type include short chain alkyl, cycloalkyl, mixed heteroatom alkyl, mixed heteroatom cycloalkyl, one or more short chain heteroatomic and one or more short chain heterocyclic. These internucleoside linkages include but are not limited to siloxane, sulfide, sulfoxide, sulfone, acetyl, formacetyl, thioformacetyl, methylene formacetyl, thioformacetyl, alkeneyl, sulfamate; methyleneimino, methylenehydrazino, sulfonate, sulfonamide, amide and others having mixed N, O, S and CH₂ component parts. In addition to the modifications described above, the nucleosides of the oligomeric compounds of the invention can have a variety of other modifications. Additional nucleosides amenable to the present invention having altered base moieties and or altered sugar moieties are disclosed in U.S. Pat. No. 3,687,808 and PCT application PCT/US89/02323.

For nucleotides that are incorporated into oligonucleotides of the invention, these nucleotides can have sugar portions that correspond to naturally occurring sugars or modified sugars. Representative modified sugars include carbocyclic or acyclic sugars, sugars having substituent groups at one or more of their 2′, 3′ or 4′ positions and sugars having substituents in place of one or more hydrogen atoms of the sugar.

Altered base moieties or altered sugar moieties also include other modifications consistent with the spirit of this invention. Such oligomeric compounds are best described as being structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified oligonucleotides. All such oligomeric compounds are comprehended by this invention so long as they function effectively to mimic the structure or function of a desired RNA or DNA oligonucleotide strand.

A class of representative base modifications include tricyclic cytosine analog, termed “G clamp” (Lin, et at., J. Am. Chem. Soc. 1998, 120, 8531). This analog can form four hydrogen bonds with a complementary guanine (G) by simultaneously recognizing the Watson-Crick and Hoogsteen faces of the targeted G. This G clamp modification when incorporated into phosphorothioate oligomeric compounds, dramatically enhances potencies as measured by target reduction in cell culture. The oligomeric compounds of the invention also can include phenoxazine-substituted bases of the type disclosed by Flanagan, et al., Nat. Biotechnol. 1999, 17(1), 48-52.

The oligomeric compounds in accordance with this invention comprise from about 8 to about 80 monomeric subunits (i.e. from about 8 to about 80 linked nucleosides). One of ordinary skill in the art will appreciate that the invention embodies oligomeric compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 12 to 50 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 13 to 80 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 15 to 30 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 70 to 450 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449 or 450 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 110 to 430 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429 or 430 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 110 to 280 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279 or 280 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 50 to 110 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 70, 71 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 or 110 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 60 to 80 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 15 to 49 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 17 to 25 subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 17, 18, 19, 20, 21, 22, 23, 24 or 25 subunits in length, or any range therewithin.

In accordance with the present invention, oligomeric compounds designed to mimic pri-miRNAs are from about 70 to about 450 monomeric subunits in length, or from about 110 to 430 subunits in length. Oligomeric compounds of the invention designed to mimic pre-miRNAs are from about 50 to about 110 monomeric subunits in length, or from about 60 to about 80 subunits in length. Oligomeric compounds of the invention designed to mimic mature miRNAs are from about 17 to about 25 monomeric subunits in length, and can be single- or double-stranded with either or both strands comprising from about 17 to about 25 subunits.

As used herein, the term “about” means±5% of the variable thereafter.

The size or length of any oligomeric compound of the present invention, within any range cited herein, can be determined as follows:

Let R(m, n+m−1) be a region from a target nucleobase sequence, where “n” is the 5′-most nucleobase position of the region, where “n+m−1” is the 3′-most nucleobase position of the region and where “m” is the length of the region. A set “S(m)”, of regions of length “m” is defined as the regions where n ranges from 1 to L−m+1, where L is the length of the target nucleic acid sequence and L>m. A set, “A”, of all regions can be constructed as a union of the sets of regions for each length from where m is greater than or equal to a lower limit of any recited range (8 in this example) and is less than or equal to the upper limit of any recited range (80 in this example).

This set of regions can be represented using the following mathematical notation:

$A = {{{\bigcup\limits_{m}{{S(m)}\mspace{14mu}{where}\mspace{14mu} m}} \in N}❘{8 \leq m \leq 80}}$

and S(m)={R _(n,n+m−1) |nε{1,2,3, . . . , L−m+1}}

where the mathematical operator | indicates “such that”,

where the mathematical operator ε indicates “a member of a set” (e.g. yεZ indicates that element y is a member of set Z),

where x is a variable,

where N indicates all natural numbers, defined as positive integers,

and where the mathematical operator ∪ indicates “the union of sets”.

For example, the set of regions for m equal to 8, 20 and 80 can be constructed in the following manner. The set of regions, each 8 monomeric subunits in length, S(m=8), in a target nucleic acid sequence 100 subunits in length (L=100), beginning at position 1 (n=1) of the target nucleic acid sequence, can be created using the following expression: S(8)={R _(1,8) |nε{1,2,3, . . . ,93}} and describes the set of regions comprising nucleobases 1-8, 2-9, 3-10, 4-11, 5-12, 6-13, 7-14, 8-15, 9-16, 10-17, 11-18, 12-19, 13-20, 14-21, 15-22, 16-23, 17-24, 18-25, 19-26, 20-27, 21-28, 22-29, 23-30, 24-31, 25-32, 26-33, 27-34, 28-35, 29-36, 30-37, 31-38, 32-39, 33-40, 34-41, 35-42, 36-43, 37-44, 38-45, 39-46, 40-47, 41-48, 42-49, 43-50, 44-51, 45-52, 46-53, 47-54, 48-55, 49-56, 50-57, 51-58, 52-59, 53-60, 54-61, 55-62, 56-63, 57-64, 58-65, 59-66, 60-67, 61-68, 62-69, 63-70, 64-71, 65-72, 66-73, 67-74, 68-75, 69-76, 70-77, 71-78, 72-79, 73-80, 74-81, 75-82, 76-83, 77-84, 78-85, 79-86, 80-87, 81-88, 82-89, 83-90, 84-91, 85-92, 86-93, 87-94, 88-95, 89-96, 90-97, 91-98, 92-99, 93-100.

An additional set for regions 20 monomeric subunits in length, in a target sequence 100 subunits in length, beginning at position 1 of the target nucleic acid sequence, can be described using the following expression: S(20)={R _(1,20) |nε{1,2,3, . . . ,81}} and describes the set of regions comprising nucleobases 1-20, 2-21, 3-22, 4-23, 5-24, 6-25, 7-26, 8-27, 9-28, 10-29, 11-30, 12-31, 13-32, 14-33, 15-34, 16-35, 17-36, 18-37, 19-38, 20-39, 21-40, 22-41, 23-42, 24-43, 25-44, 26-45, 27-46, 28-47, 29-48, 30-49, 31-50, 32-51, 33-52, 34-53, 35-54, 36-55, 37-56, 38-57, 39-58, 40-59, 41-60, 42-61, 43-62, 44-63, 45-64, 46-65, 47-66, 48-67, 49-68, 50-69, 51-70, 52-71, 53-72, 54-73, 55-74, 56-75, 57-76, 58-77, 59-78, 60-79, 61-80, 62-81, 63-82, 64-83, 65-84, 66-85, 67-86, 68-87, 69-88, 70-89, 71-90, 72-91, 73-92, 74-93, 75-94, 76-95, 77-96, 78-97, 79-98, 80-99, 81-100.

An additional set for regions 80 monomeric subunits in length, in a target sequence 100 subunits in length, beginning at position 1 of the target nucleic acid sequence, can be described using the following expression: S(80)={R _(1,80) |nε{1,2,3, . . . ,21}} and describes the set of regions comprising nucleobases 1-80, 2-81, 3-82, 4-83, 5-84, 6-85, 7-86, 8-87, 9-88, 10-89, 11-90, 12-91, 13-92, 14-93, 15-94, 16-95, 17-96, 18-97, 19-98, 20-99, 21-100.

The union of these aforementioned example sets and other sets for lengths from 10 to 19 and 21 to 79 can be described using the mathematical expression

$A = {\bigcup\limits_{m}{S(m)}}$ where ∪ represents the union of the sets obtained by combining all members of all sets.

Thus, in this example, A would include regions 1-8, 2-9, 3-10 . . . 93-100, 1-20, 2-21, 3-22 . . . 81-100, 1-80, 2-81, 3-82 . . . 21-100.

The mathematical expressions described herein define all possible target regions in a target nucleic acid sequence of any length L, where the region is of length m, and where m is greater than or equal to the lower limit and less than or equal to the upper limit of monomeric units, and where m is less than L, and where n is less than L−m+1.

In the context of this invention, “hybridization” means the pairing of complementary strands of oligomeric compounds. In the present invention, the mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.

An oligomeric compound of the invention is “specifically hybridizable” when association of the compound with the target nucleic acid interferes with the normal function of the target nucleic acid to alter the activity, disrupt the function, or modulate the level of the target nucleic acid, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target nucleic acid sequences under conditions in which specific hybridization is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under standard assay conditions in the case of in vitro assays.

In the present invention the phrase “stringent hybridization conditions” or “stringent conditions” refers to conditions under which an oligomeric compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will vary with different circumstances and in the context of this invention; “stringent conditions” under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated. One having ordinary skill in the art will understand variability in the experimental protocols and be able to determine when conditions are optimal for stringent hybridization with minimal non-specific hybridization events.

“Complementary,” as used herein, refers to the capacity for precise pairing of two monomeric subunits regardless of where in the oligomeric compound or target nucleic acid the two are located. For example, if a monomeric subunit at a certain position of an oligomeric compound is capable of hydrogen bonding with a monomeric subunit at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligomeric compound and the target nucleic acid is considered to be a complementary position. The oligomeric compound and the target nucleic acid are “substantially complementary” to each other when a sufficient number of complementary positions in each molecule are occupied by monomeric subunits that can hydrogen bond with each other. Thus, the term “substantially complementary” is used to indicate a sufficient degree of precise pairing over a sufficient number of monomeric subunits such that stable and specific binding occurs between the oligomeric compound and a target nucleic acid.

Generally, an oligomeric compound is “antisense” to a target nucleic acid when, written in the 5′ to 3′ direction, it comprises the reverse complement of the corresponding region of the target nucleic acid. “Antisense compounds” are also often defined in the art to comprise the further limitation of, once hybridized to a target, being able to induce or trigger a reduction in target gene expression.

It is understood in the art that the sequence of the oligomeric compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligomeric compound may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization (e.g., a bulge, a loop structure or a hairpin structure).

In some embodiments of the invention, the oligomeric compounds comprise at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, or at least 85% sequence complementarity to a target region within the target nucleic acid. In other embodiments of the invention, the oligomeric compounds comprise at least 90% sequence complementarity to a target region within the target nucleic acid. In other embodiments of the invention, the oligomeric compounds comprise at least 95% or at least 99% sequence complementarity to a target region within the target nucleic acid. For example, an oligomeric compound in which 18 of 20 nucleobases of the oligomeric compound are complementary to a target sequence would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an oligomeric compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an oligomeric compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

In some embodiments of the invention, the oligomeric compounds act as mimics or replacements for small non-coding RNAs. In this case, the oligomeric compounds of the invention can comprise at least 70% sequence identity to a small non-coding RNA or a region thereof. In some embodiments the oligomeric compounds of the invention can comprise at least 90% sequence identity and in some embodiments can comprise at least 95% sequence identity to a small non-coding RNA or a region thereof.

“Targeting” an oligomeric compound to a particular nucleic acid molecule, in the context of this invention, can be a multistep process. The process usually begins with the identification of a target nucleic acid whose levels, expression or function is to be modulated. This target nucleic acid may be, for example, a mRNA transcribed from a cellular gene whose expression is associated with a particular disorder or disease state, a small non-coding RNA or its precursor, or a nucleic acid molecule from an infectious agent.

The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the interaction to occur such that the desired effect, e.g., modulation of levels, expression or function, will result. Within the context of the present invention, the term “region” is defined as a portion of the target nucleic acid having at least one identifiable sequence, structure, function, or characteristic. Within regions of target nucleic acids are segments. “Segments” are defined as smaller or sub-portions of regions within a target nucleic acid. “Sites,” as used in the present invention, are defined as specific positions within a target nucleic acid. The terms region, segment, and site can also be used to describe an oligomeric compound of the invention such as for example a gapped oligomeric compound having three separate segments.

Targets of the present invention include both coding and non-coding nucleic acid sequences. For coding nucleic acid sequences, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon.” A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding a nucleic acid target, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon. Consequently, the “start codon region” (or “translation initiation codon region”) and the “stop codon region” (or “translation termination codon region”) are all regions which may be targeted effectively with the oligomeric compounds of the present invention.

The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Within the context of the present invention, a further suitable region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.

Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene), and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA (or corresponding nucleotides on the gene). The 5′ cap site of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap site. It is also suitable to target the 5′ cap region.

Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. Targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, may also be particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also target sites. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts.” It is also known that introns can be effectively targeted using oligomeric compounds targeted to, precursor molecules for example, pre-mRNA.

It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants.” More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequences.

Upon excision of one or more exon or intron regions, or portions thereof, during splicing, pre-mRNA variants produce smaller “mRNA variants.” Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants.” If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as “alternative start variants” of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or mRNA. One specific type of alternative stop variant is the “polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the “polyA stop signals” by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. Within the context of the invention, the types of variants described herein are also target nucleic acids.

Certain non-coding RNA genes are known to produce functional RNA molecules with important roles in diverse cellular processes. Such non-translated, non-coding RNA molecules can include ribosomal RNAs, tRNAs, snRNAs, snoRNAs, tncRNAs, rasiRNAs, short hairpin RNAs (shRNAs), short temporal RNAs (stRNAs), short hairpin RNAs (shRNAs), siRNAs, miRNAs and smnRNAs. These non-coding RNA genes and their products are also suitable targets of the compounds of the invention. Such cellular processes include transcriptional regulation, translational regulation, developmental timing, viral surveillance, immunity, chromosome maintenance, ribosomal structure and function, gene imprinting, subcellular compartmentalization, pre-mRNA splicing, and guidance of RNA modifications. RNA-mediated processes are now also believed to direct heterochromatin formation, genome rearrangements, cellular differentiation and DNA elimination.

A total of 201 different expressed RNA sequences potentially encoding novel small non-messenger species (smnRNAs) has been identified from mouse brain cDNA libraries. Based on sequence and structural motifs, several of these have been assigned to the snoRNA class of nucleolar localized molecules known to act as guide RNAs for rRNA modification, whereas others are predicted to direct modification within the U2, U4, or U6 small nuclear RNAs (snRNAs). Some of these newly identified smnRNAs remained unclassified and have no identified RNA targets. It was suggested that some of these RNA species may have novel functions previously unknown for snoRNAs, namely the regulation of gene expression by binding to and/or modifying mRNAs or their precursors via their antisense elements (Huttenhofer et al., Embo J., 2001, 20, 2943-2953). Therefore, these smRNAs are also suitable targets for the compounds of the present invention.

The locations on the target nucleic acid to which compounds and compositions of the invention hybridize are herein referred to as “suitable target segments.” As used herein the term “suitable target segment” is defined as at least an 8-nucleobase portion of a target region to which oligomeric compound is targeted.

Once one or more targets, target regions, segments or sites have been identified, oligomeric compounds are designed to be sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect. The desired effect may include, but is not limited to modulation of the levels, expression or function of the target.

In accordance with the present invention, a series of single stranded oligomeric compounds can be designed to target or mimic one or more specific small non-coding RNAs. These oligomeric compounds can be of a specified length, for example from 8 to 80, 12 to 50, 13 to 80, 15 to 30, 70 to 450, 110 to 430, 110 to 280, 50 to 110, 60 to 80, 15 to 49, 17 to 25 or 19 to 23 nucleotides long and have one or more modifications.

In accordance with one embodiment of the invention, a series of double-stranded oligomeric compounds (duplexes) comprising, as the antisense strand, the single-stranded oligomeric compounds of the present invention, and the fully or partially complementary sense strand, can be designed to modulate the levels, expression or function of one or more small non-coding RNAs or small non-coding RNA targets. One or both termini of the duplex strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the duplex may be designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in one embodiment, both strands of the duplex would be complementary over the central region of the duplex, each having overhangs at one or both termini.

For the purposes of this invention, the combination of an antisense strand and a sense strand, each of which can be of a specified length, for example from 8 to 80, 12 to 50, 13 to 80, 15 to 30, 15 to 49, 17 to 25 or 19 to 23 subunits long, is identified as a complementary pair of oligomeric compounds. This complementary pair of oligonucleotides can include additional nucleotides on either of their 5′ or 3′ ends. They can include other molecules or molecular structures on their 3′ or 5′ ends, such as a phosphate group on the 5′ end, or non-nucleic acid moieties conjugated to either terminus of either strand or both strands. One group of compounds of the invention includes a phosphate group on the 5′ end of the antisense strand compound. Other compounds also include a phosphate group on the 5′ end of the sense strand compound. Some compounds include additional nucleotides such as a two base overhang on the 3′ end as well as those lacking overhangs.

For example, a complementary pair of oligomeric compounds may comprise an antisense strand oligomeric compound having the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO:2181), having a two-nucleobase overhang of deoxythymidine (dT) and its complement sense strand. This complementary pair of oligomeric compounds would have the following structure:

In some embodiments, a single-stranded oligomeric compound may be designed comprising the antisense portion as a first region and the sense portion as a second region. The first and second regions can be linked together by either a nucleotide linker (a string of one or more nucleotides that are linked together in a sequence) or by a non-nucleotide linker region or by a combination of both a nucleotide and non-nucleotide structure. In any of these structures, the oligomeric compound, when folded back on itself, would form at least a partially complementary structure at least between a portion of the first region, the antisense portion, and a portion of the second region, the sense portion.

In one embodiment, the invention includes an oligomeric compound/protein composition. This composition has both an oligomeric compound component and a protein component. The oligomeric compound component comprises at least one oligomeric compound, either the antisense or the sense oligomeric compound but preferably the antisense oligomeric compound (the oligomeric compound that is antisense to the target nucleic acid). The protein component of the composition comprises at least one protein that forms a portion of the RNA-induced silencing complex, i.e., the RISC complex. The oligomeric compound component can also comprise both antisense and sense strand oligomeric compounds.

RISC is a ribonucleoprotein complex that contains proteins of the Argonaute family of proteins. While not wishing to be bound by theory, it is believed that the Argonaute proteins are a class of proteins, some of which have been shown to contain a PAZ and/or a Piwi domain and that have been implicated in processes previously linked to posttranscriptional silencing. The Argonaute family of proteins includes, but depending on species, is not necessary limited to e1F2C1 and e1F2C2. It is also believed that at least the antisense strand of double-stranded compounds shown to act as siRNAs is bound to one of the protein components that form the RISC complex, and that the RISC complex interacts with the ribosomes or polyribosome complexes which may contain small non-coding RNA molecules amenable to targeting with the oligomeric compounds of the present invention. Consequently, one embodiment of the invention includes oligomeric compounds that mimic RNA components of the RISC complex.

In one embodiment, the oligomeric compounds of the invention are designed to exert their modulatory effects via mimicking or targeting small non-coding RNAs associated with cellular factors such as transporters or chaperones. These cellular factors can be protein, lipid or carbohydrate based and can have structural or enzymatic functions that may or may not require the complexation of one or more metal ions.

Furthermore, the oligomeric compounds of the invention can have one or more moieties bound or conjugated, which facilitates the active or passive transport, localization, or compartmentalization of the oligomeric compound. Cellular localization includes, but is not limited to, localization to within the nucleus, the nucleolus, or the cytoplasm. Compartmentalization includes, but is not limited to, any directed movement of the oligonucleotides of the invention to a cellular compartment including the nucleus, nucleolus, mitochondrion, or imbedding into a cellular membrane.

In some embodiments of the invention, the oligomeric compounds are designed to exert their modulatory effects via mimicking or targeting small non-coding RNAs associated with cellular factors that affect gene expression, more specifically those involved in RNA or DNA modifications. These modifications include, but are not limited to, posttranscriptional or chromosomal modifications such as methylation, acetylation, pseudouridylation or amination.

Furthermore, the oligomeric compounds of the invention comprise one or more conjugate moieties which facilitate posttranscriptional modification.

The oligomeric compounds of the invention may be in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops. Once introduced to a system, the oligomeric compounds of the invention may elicit the action of one or more enzymes or proteins to effect modulation of the levels, expression or function of the target nucleic acid.

One non-limiting example of such a protein is the Drosha RNase III enzyme. Drosha is a nuclear enzyme that processes long primary RNA transcripts (pri-miRNAs) from approximately 70 to 450 nucleotides in length into pre-miRNAs (from about 50 to about 80 nucleotides in length) which are exported from the nucleus to encounter the human Dicer enzyme which then processes pre-miRNAs into miRNAs. It is believed that, in processing the pri-miRNA into the pre-miRNA, the Drosha enzyme cuts the pri-miRNA at the base of the mature miRNA, leaving a 2-nt 3′ overhang (Lee, et al., Nature, 2003, 425, 415-419). The 3′ two-nucleotide overhang structure, a signature of RNaseIII enzymatic cleavage, has been identified as a critical specificity determinant in targeting and maintaining small RNAs in the RNA interference pathway (Murchison, et al., Curr. Opin. Cell Biol., 2004, 16, 223-9).

A further non-limiting example involves the enzymes of the RISC complex. Use of the RISC complex to effect cleavage of RNA targets thereby greatly enhances the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.

Oligomeric compounds or compositions of the invention are used to induce potent and specific modulation of gene function through interactions with or mimicry of small non-coding RNAs that are processed by the RISC complex. These compounds include single-stranded oligomeric compounds that bind in a RISC complex, double-stranded antisense/sense pairs of oligomeric compounds, or single-stranded oligomeric compounds that include both an antisense portion and a sense portion.

General Oligomer Synthesis:

Oligomerization of modified and unmodified nucleosides is performed according to literature procedures for DNA like compounds (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/or RNA like compounds (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications of Chemically synthesized RNA in RNA:Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713) synthesis as appropriate. In addition, specific protocols for the synthesis of oligomeric compounds of the invention are illustrated in the examples below.

RNA oligomers can be synthesized by methods disclosed herein or purchased from various RNA synthesis companies such as for example Dharmacon Research Inc., (Lafayette, Colo.).

Irrespective of the particular protocol used, the oligomeric compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed.

Synthesis of Nucleoside Phosphoramidites:

The following compounds, including amidites and their intermediates were prepared as described in U.S. Pat. No. 6,426,220 and published PCT WO 02/36743; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for 5-methyl-dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimate intermediate for 5-methyl dC amidite, (5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine, 2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modified amidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate, 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate, (5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T amidite), 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine intermediate, 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidine penultimate intermediate, (5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite), (5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A amdite), (5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylaminooxyethyl) nucleoside amidites, 2′-(Dimethylaminooxyethoxy) nucleoside amidites, 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine, 2′-O-((2-phthalimidoxy)ethyl)-5′-t-butyldiphenylsilyl-5-methyluridine, 5′-O-tert-butyldiphenylsilyl-2′-O-((2-formadoximinooxy)ethyl)-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O—(N,N dimethylaminooxyethyl)-5-methyluridine, 2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-((2-cyanoethyl)-N,N-diisopropylphosphoramidite), 2′-(Aminooxyethoxy) nucleoside amidites, N²-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-((2-cyanoethyl)-N,N-diisopropylphosphoramidite), 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites, 2′-O-(2(2-N,N-dimethylaminoethoxy)ethyl)-5-methyl uridine, 5′-O-dimethoxytrityl-2′-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methyl uridine and 5′-O-Dimethoxytrityl-2′-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Oligonucleotide and Oligonucleoside Synthesis:

Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligonucleotides were recovered by precipitating with >3 volumes of ethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporated by reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.

Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Oligonucleosides: Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone oligomeric compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

RNA Synthesis:

In general, RNA synthesis chemistry is based on the selective incorporation of various protecting groups at strategic intermediary reactions. Although one of ordinary skill in the art will understand the use of protecting groups in organic synthesis, a useful class of protecting groups includes silyl ethers. In particular bulky silyl ethers are used to protect the 5′-hydroxyl in combination with an acid-labile orthoester protecting group on the 2′-hydroxyl. This set of protecting groups is then used with standard solid-phase synthesis technology. It is important to lastly remove the acid labile orthoester protecting group after all other synthetic steps. Moreover, the early use of the silyl protecting groups during synthesis ensures facile removal when desired, without undesired deprotection of 2′ hydroxyl.

Following this procedure for the sequential protection of the 5′-hydroxyl in combination with protection of the 2′-hydroxyl by protecting groups that are differentially removed and are differentially chemically labile, RNA oligonucleotides were synthesized.

RNA oligonucleotides are synthesized in a stepwise fashion. Each nucleotide is added sequentially (3′- to 5′-direction) to a solid support-bound oligonucleotide. The first nucleoside at the 3′-end of the chain is covalently attached to a solid support. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are added, coupling the second base onto the 5′-end of the first nucleoside. The support is washed and any unreacted 5′-hydroxyl groups are capped with acetic anhydride to yield 5′-acetyl moieties. The linkage is then oxidized to the more stable and ultimately desired P(V) linkage. At the end of the nucleotide addition cycle, the 5′-silyl group is cleaved with fluoride. The cycle is repeated for each subsequent nucleotide.

Following synthesis, the methyl protecting groups on the phosphates are cleaved in 30 minutes utilizing 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂) in DMF. The deprotection solution is washed from the solid support-bound oligonucleotide using water. The support is then treated with 40% methylamine in water for 10 minutes at 55° C. This releases the RNA oligonucleotides into solution, deprotects the exocyclic amines, and modifies the 2′-groups. The oligonucleotides can be analyzed by anion exchange HPLC at this stage.

The 2′-orthoester groups are the last protecting groups to be removed. The ethylene glycol monoacetate orthoester protecting group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is one example of a useful orthoester protecting group which, has the following important properties. It is stable to the conditions of nucleoside phosphoramidite synthesis and oligonucleotide synthesis. However, after oligonucleotide synthesis the oligonucleotide is treated with methylamine which not only cleaves the oligonucleotide from the solid support but also removes the acetyl groups from the orthoesters. The resulting 2-ethyl-hydroxyl substituents on the orthoester are less electron withdrawing than the acetylated precursor. As a result, the modified orthoester becomes more labile to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is approximately 10 times faster after the acetyl groups are removed. Therefore, this orthoester possesses sufficient stability in order to be compatible with oligonucleotide synthesis and yet, when subsequently modified, permits deprotection to be carried out under relatively mild aqueous conditions compatible with the final RNA oligonucleotide product.

Additionally, methods of RNA synthesis are well known in the art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand., 1990, 44, 639-641; Reddy, M. P., et al., Tetrahedrom Lett, 1994, 25, 4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2315-2331).

The present invention is also useful for the preparation of oligomeric compounds incorporating at least one 2′-O-protected nucleoside. After incorporation and appropriate deprotection the 2′-O-protected nucleoside will be converted to a ribonucleoside at the position of incorporation. The number and position of the 2-ribonucleoside units in the final oligomeric compound can vary from one at any site or the strategy can be used to prepare up to a full 2′-OH modified oligomeric compound. All 2′-O-protecting groups amenable to the synthesis of oligomeric compounds are included in the present invention.

In general a protected nucleoside is attached to a solid support by for example a succinate linker. Then the oligonucleotide is elongated by repeated cycles of deprotecting the 5′-terminal hydroxyl group, coupling of a further nucleoside unit, capping and oxidation (alternatively sulfurization). In a more frequently used method of synthesis the completed oligonucleotide is cleaved from the solid support with the removal of phosphate protecting groups and exocyclic amino protecting groups by treatment with an ammonia solution. Then a further deprotection step is normally required for the more specialized protecting groups used for the protection of 2′-hydroxyl groups which will give the fully deprotected oligonucleotide.

A large number of 2′-O-protecting groups have been used for the synthesis of oligoribonucleotides but over the years more effective groups have been discovered. The key to an effective 2′-O-protecting group is that it is capable of selectively being introduced at the 2′-O-position and that it can be removed easily after synthesis without the formation of unwanted side products. The protecting group also needs to be inert to the normal deprotecting, coupling, and capping steps required for oligoribonucleotide synthesis. Some of the protecting groups used initially for oligoribonucleotide synthesis included tetrahydropyran-1-yl and 4-methoxytetrahydropyran-4-yl. These two groups are not compatible with all 5′-O-protecting groups so modified versions were used with 5′-DMT groups such as 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp). Reese has identified a number of piperidine derivatives (like Fpmp) that are useful in the synthesis of oligoribonucleotides including 1-((chloro-4-methyl)phenyl)-4′-methoxypiperidin-4-yl (Reese et al, Tetrahedron Lett., 1986, (27), 2291). Another approach was to replace the standard 5′-DMT (dimethoxytrityl) group with protecting groups that were removed under non-acidic conditions such as levulinyl and 9-fluorenylmethoxycarbonyl. Such groups enable the use of acid labile 2′-protecting groups for oligoribonucleotide synthesis. Another more widely used protecting group initially used for the synthesis of oligoribonucleotides was the t-butyldimethylsilyl group (Ogilvie et al, Tetrahedron Lett., 1974, 2861; Hakimelahi et al, Tetrahedron Lett., 1981, (22), 2543; and Jones et at., J. Chem. Soc. Perkin I., 2762). The 2′-O-protecting groups can require special reagents for their removal such as for example the t-butyldimethylsilyl group is normally removed after all other cleaving/deprotecting steps by treatment of the oligomeric compound with tetrabutylammonium fluoride (TBAF).

One group of researchers examined a number of 2′-O-protecting groups (Pitsch, S., Chimia, 2001, (55), 320-324.) The group examined fluoride labile and photolabile protecting groups that are removed using moderate conditions. One photolabile group that was examined was the (2-(nitrobenzyl)oxy)methyl (nbm) protecting group (Schwartz et al, Bioorg. Med. Chem. Lett., 1992, (2), 1019.) Other groups examined included a number structurally related formaldehyde acetal-derived, 2′-O-protecting groups. Also prepared were a number of related protecting groups for preparing 2′-O-alkylated nucleoside phosphoramidites including 2′-O-((triisopropylsilyl)oxy)methyl (2′-O—CH₂—O—Si(iPr)₃, TOM). One 2′-O-protecting group that was prepared to be used orthogonally to the TOM group was 2′-O-((R)-1-(2-nitrophenyl)ethyloxy)methyl) ((R)-mnbm).

Another strategy using a fluoride labile 5′-O-protecting group (non-acid labile) and an acid labile 2′-O-protecting group has been reported (Scaringe, Stephen A., Methods, 2001, (23) 206-217). A number of possible silyl ethers were examined for 5′-O-protection and a number of acetals and orthoesters were examined for 2′-O-protection. The protection scheme that gave the best results was 5′-O-silyl ether-2′-ACE (5′-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether (DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). This approach uses a modified phosphoramidite synthesis approach in that some different reagents are required that are not routinely used for RNA/DNA synthesis.

Although a lot of research has focused on the synthesis of oligoribonucleotides the main RNA synthesis strategies that are presently being used commercially include 5′-O-DMT-2′-O-t-butyldimethylsilyl (TBDMS), 5′-O-DMT-2′-O-(1(2-fluorophenyl)-4-methoxypiperidin-4-yl) (FPMP), 2′-O-((triisopropylsilyl)oxy)methyl (2′-O—CH₂—O—Si(iPr)₃ (TOM), and the 5′-O-silyl ether-2′-ACE (5′-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether (DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). A current list of some of the major companies currently offering RNA products include Pierce Nucleic Acid Technologies, Dharmacon Research Inc., Ameri Biotechnologies Inc., and Integrated DNA Technologies, Inc. One company, Princeton Separations, is marketing an RNA synthesis activator advertised to reduce coupling times especially with TOM and TBDMS chemistries. Such an activator would also be amenable to the present invention.

The structures corresponding to these protecting groups are shown below.

TBDMS=5′-O-DMT-2′-O-t-butyldimethylsilyl;

TOM=2′-O-((triisopropylsilyl)oxy)methyl;

DOD/ACE=(5′-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether-2′-O-bis(2-acetoxyethoxy)methyl

FPMP=5′-O-DMT-2′-O-(1 (2-fluorophenyl)-4-methoxypiperidin-4-yl)

All of the aforementioned RNA synthesis strategies are amenable to the present invention. Strategies that would be a hybrid of the above e.g. using a 5′-protecting group from one strategy with a 2′-O-protecting from another strategy is also amenable to the present invention.

The preparation of ribonucleotides and oligomeric compounds having at least one ribonucleoside incorporated and all the possible configurations falling in between these two extremes are encompassed by the present invention. The corresponding oligomeric compounds can be hybridized to further oligomeric compounds including oligoribonucleotides having regions of complementarity to form double-stranded (duplexed) oligomeric compounds.

The methods of preparing oligomeric compounds of the present invention can also be applied in the areas of drug discovery and target validation.

Oligonucleotide Isolation:

After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH₄OAc with >3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis was determined by the ratio of correct molecular weight relative to the −16 amu product (+/−32 +/−48). For some studies oligonucleotides were purified by HPLC, as described by Chiang et at., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Oligonucleotide Synthesis—96 Well Plate Format:

Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected with concentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Oligonucleotide Analysis—96-Well Plate Format:

The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the oligomeric compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the oligomeric compounds on the plate were at least 85% full length.

For double-stranded compounds of the invention, once synthesized, the complementary strands are annealed. The single strands are aliquoted and diluted to a concentration of 50 μM. Once diluted, 30 μL of each strand is combined with 15 μL of a 5× solution of annealing buffer. The final concentration of the buffer is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume is 75 μL. This solution is incubated for 1 minute at 90° C. and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37° C. at which time the double-stranded compounds are used in experimentation. The final concentration of the duplexed compound is 20 μM. This solution can be stored frozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the double-stranded compounds are evaluated for their ability to modulate target levels, expression or function. When cells reach 80% confluency, they are treated with synthetic double-stranded compounds comprising at least one oligomeric compound of the invention. For cells grown in 96-well plates, wells are washed once with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM™-1 containing 12 μg/mL LIPOFECTIN™ (Invitrogen Corporation, Carlsbad, Calif.) and the desired double stranded compound at a final concentration of 200 nM. After 5 hours of treatment, the medium is replaced with fresh medium. Cells are harvested 16 hours after treatment, at which time RNA is isolated and target reduction measured by real-time RT-PCR.

Specific examples of oligomeric compounds useful in this invention include oligonucleotides containing modified e.g. non-naturally occurring internucleoside linkages. As defined in this specification, oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom and internucleoside linkages that do not have a phosphorus atom. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

In the C. elegans system, modification of the internucleotide linkage (phosphorothioate) did not significantly interfere with RNAi activity. Based on this observation, it is suggested that certain oligomeric compounds of the invention can also have one or more modified internucleoside linkages. A suitable phosphorus-containing modified internucleoside linkage is the phosphorothioate internucleoside linkage.

Modified oligonucleotide backbones (internucleoside linkages) containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. No. 3,687,808; U.S. Pat. No. 4,469,863; U.S. Pat. No. 4,476,301; U.S. Pat. No. 5,023,243; U.S. Pat. No. 5,177,196; U.S. Pat. No. 5,188,897; U.S. Pat. No. 5,264,423; U.S. Pat. No. 5,276,019; U.S. Pat. No. 5,278,302; U.S. Pat. No. 5,286,717; U.S. Pat. No. 5,321,131; U.S. Pat. No. 5,399,676; U.S. Pat. No. 5,405,939; U.S. Pat. No. 5,453,496; U.S. Pat. No. 5,455,233; U.S. Pat. No. 5,466,677; U.S. Pat. No. 5,476,925; U.S. Pat. No. 5,519,126; U.S. Pat. No. 5,536,821; U.S. Pat. No. 5,541,306; U.S. Pat. No. 5,550,111; U.S. Pat. No. 5,563,253; U.S. Pat. No. 5,571,799; U.S. Pat. No. 5,587,361; U.S. Pat. No. 5,194,599; U.S. Pat. No. 5,565,555; U.S. Pat. No. 5,527,899; U.S. Pat. No. 5,721,218; U.S. Pat. No. 5,672,697 and U.S. Pat. No. 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

In other embodiments of the invention, oligomeric compounds have one or more phosphorothioate and/or heteroatom internucleoside linkages, in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— (known as a methylene (methylimino) or MMI backbone), —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— (wherein the native phosphodiester internucleotide linkage is represented as —O—P(═O)(OH)—O—CH₂—). The MMI type internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,489,677. Amide internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,602,240.

Modified oligonucleotide backbones (internucleoside linkages) that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. No. 5,034,506; U.S. Pat. No. 5,166,315; U.S. Pat. No. 5,185,444; U.S. Pat. No. 5,214,134; U.S. Pat. No. 5,216,141; U.S. Pat. No. 5,235,033; U.S. Pat. No. 5,264,562; U.S. Pat. No. 5,264,564; U.S. Pat. No. 5,405,938; U.S. Pat. No. 5,434,257; U.S. Pat. No. 5,466,677; U.S. Pat. No. 5,470,967; U.S. Pat. No. 5,489,677; U.S. Pat. No. 5,541,307; U.S. Pat. No. 5,561,225; U.S. Pat. No. 5,596,086; U.S. Pat. No. 5,602,240; U.S. Pat. No. 5,610,289; U.S. Pat. No. 5,602,240; U.S. Pat. No. 5,608,046; U.S. Pat. No. 5,610,289; U.S. Pat. No. 5,618,704; U.S. Pat. No. 5,623,070; U.S. Pat. No. 5,663,312; U.S. Pat. No. 5,633,360; U.S. Pat. No. 5,677,437; U.S. Pat. No. 5,792,608; U.S. Pat. No. 5,646,269 and U.S. Pat. No. 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

Another group of oligomeric compounds amenable to the present invention includes oligonucleotide mimetics. The term mimetic as it is applied to oligonucleotides is intended to include oligomeric compounds wherein only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with novel groups, replacement of only the furanose ring is also referred to in the art as being a sugar surrogate. The heterocyclic base moiety or a modified heterocyclic base moiety is maintained for hybridization with an appropriate target nucleic acid. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA oligomeric compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA oligomeric compounds include, but are not limited to, U.S. Pat. No. 5,539,082; U.S. Pat. No. 5,714,331; and U.S. Pat. No. 5,719,262, each of which is herein incorporated by reference. Teaching of PNA oligomeric compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.

PNA has been modified to incorporate numerous modifications since the basic PNA structure was first prepared. The basic structure is shown below:

wherein

Bx is a heterocyclic base moiety;

T₄ is hydrogen, an amino protecting group, —C(O)R₅, substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group, a reporter group, a conjugate group, a D or L α-amino acid linked via the α-carboxyl group or optionally through the ω-carboxyl group when the amino acid is aspartic acid or glutamic acid or a peptide derived from D, L or mixed D and L amino acids linked through a carboxyl group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;

T₅ is —OH, —N(Z₁)Z₂, R₅, D or L α-amino acid linked via the α-amino group or optionally through the ω-amino group when the amino acid is lysine or ornithine or a peptide derived from D, L or mixed D and L amino acids linked through an amino group, a chemical functional group, a reporter group or a conjugate group;

Z₁ is hydrogen, C₁-C₆ alkyl, or an amino protecting group;

Z₂ is hydrogen, C₁-C₆ alkyl, an amino protecting group, —C(═O)—(CH₂)_(n)-J-Z₃, a D or L α-amino acid linked via the α-carboxyl group or optionally through the ω-carboxyl group when the amino acid is aspartic acid or glutamic acid or a peptide derived from D, L or mixed D and L amino acids linked through a carboxyl group;

Z₃ is hydrogen, an amino protecting group, —C₁-C₆ alkyl, —C(═O)—CH₃, benzyl, benzoyl, or —(CH₂)_(n)—N(H)Z₁;

each J is O, S or NH;

R₅ is a carbonyl protecting group; and

n is from 2 to about 450.

Another class of oligonucleotide mimetic that has been studied is based on linked morpholino units (morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring. A number of linking groups have been reported that link the morpholino monomeric units in a morpholino nucleic acid. A suitable class of linking groups have been selected to give a non-ionic oligomeric compound. The non-ionic morpholino-based oligomeric compounds are less likely to have undesired interactions with cellular proteins. Morpholino-based oligomeric compounds are non-ionic mimics of oligonucleotides which are less likely to form undesired interactions with cellular proteins (Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based oligomeric compounds are disclosed in U.S. Pat. No. 5,034,506, issued Jul. 23, 1991. The morpholino class of oligomeric compounds have been prepared having a variety of different linking groups joining the monomeric subunits.

Morpholino nucleic acids have been prepared having a variety of different linking groups (L₂) joining the monomeric subunits. The basic formula is shown below:

wherein

T₁ is hydroxyl or a protected hydroxyl;

T₅ is hydrogen or a phosphate or phosphate derivative;

L₂ is a linking group; and

n is from 2 to about 450.

Another class of oligonucleotide mimetic is referred to as cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in an DNA/RNA molecule is replaced with a cyclohenyl ring. CeNA DMT protected phosphoramidite monomers have been prepared and used for oligomeric compound synthesis following classical phosphoramidite chemistry. Fully modified CeNA oligomeric compounds and oligonucleotides having specific positions modified with CeNA have been prepared and studied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). In general the incorporation of CeNA monomers into a DNA chain increases its stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA and DNA complements with similar stability to the native complexes. The study of incorporating CeNA structures into natural nucleic acid structures was shown by NMR and circular dichroism to proceed with easy conformational adaptation. Furthermore the incorporation of CeNA into a sequence targeting RNA was stable to serum and able to activate E. Coli RNase resulting in cleavage of the target RNA strand.

The general formula of CeNA is shown below:

wherein

each Bx is a heterocyclic base moiety;

T₁ is hydroxyl or a protected hydroxyl;

T₂ is hydroxyl or a protected hydroxyl;

L₃ is a linking group; and

n is from 2 to about 450.

Another class of oligonucleotide mimetic (anhydrohexitol nucleic acid) can be prepared from one or more anhydrohexitol nucleosides (see, Wouters and Herdewijn, Bioorg. Med. Chem. Lett., 1999, 9, 1563-1566) and would have the general formula:

Another group of modifications includes nucleosides having sugar moieties that are bicyclic thereby locking the sugar conformational geometry. The most studied of these nucleosides is a bicyclic sugar moiety having a 4′-CH₂—O-2′ bridge. As can be seen in the structure below the 2′-O— has been linked via a methylene group to the 4′ carbon. This bridge attaches under the sugar as shown forcing the sugar ring into a locked 3′-endo conformation geometry. The ∀-L nucleoside has also been reported wherein the linkage is above the ring and the heterocyclic base is in the ∀ rather than the ∃-conformation (see U.S. Patent Application Publication No.: Application 2003/0087230). The xylo analog has also been prepared (see U.S. Patent Application Publication No.: 2003/0082807). The preferred bridge for a locked nucleic acid (LNA) is 4′—(—CH₂—)_(n)—O-2′ wherein n is 1 or 2. The literature is confusing when the term locked nucleic acid is used but in general locked nucleic acids refers to n=1, ENA™ refers to n=2 (Kaneko et al., U.S. Patent Application Publication No.: US 2002/0147332, Singh et al., Chem. Commun., 1998, 4, 455-456, also see U.S. Pat. Nos. 6,268,490 and 6,670,461 and U.S. Patent Application Publication No.: US 2003/0207841). However the term locked nucleic acids can also be used in a more general sense to describe any bicyclic sugar moiety that has a locked conformation.

ENA™ along with LNA (n=1) have been studied more than the myriad of other analogs. Oligomeric compounds incorporating LNA and ENA analogs display very high duplex thermal stabilities with complementary DNA and RNA (Tm=+3 to +10 C), stability towards 3′-exonucleolytic degradation and good solubility properties.

The basic structure of LNA showing the bicyclic ring system is shown below:

wherein

each Bx is a heterocyclic base moiety;

each L₁ is an internucleoside linking group;

T₁ is hydroxyl or a protected hydroxyl;

T₂ is hydroxyl or a protected hydroxyl, and

n is from 1 to about 80.

The conformations of LNAs determined by 2D NMR spectroscopy have shown that the locked orientation of the LNA nucleotides, both in single-stranded LNA and in duplexes, constrains the phosphate backbone in such a way as to introduce a higher population of the N-type conformation (Petersen et al., J. Mol. Recognit., 2000, 13, 44-53). These conformations are associated with improved stacking of the nucleobases (Wengel et al., Nucleosides Nucleotides, 1999, 18, 1365-1370).

LNA has been shown to form exceedingly stable LNA:LNA duplexes (Koshkin et al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNA hybridization was shown to be the most thermally stable nucleic acid type duplex system, and the RNA-mimicking character of LNA was established at the duplex level. Introduction of 3 LNA monomers (T or A) significantly increased melting points (Tm=+15/+11) toward DNA complements. The universality of LNA-mediated hybridization has been stressed by the formation of exceedingly stable LNA:LNA duplexes. The RNA-mimicking of LNA was reflected with regard to the N-type conformational restriction of the monomers and to the secondary structure of the LNA:RNA duplex.

LNAs also form duplexes with complementary DNA, RNA or LNA with high thermal affinities. Circular dichroism (CD) spectra show that duplexes involving fully modified LNA (esp. LNA:RNA) structurally resemble an A-form RNA:RNA duplex. Nuclear magnetic resonance (NMR) examination of an LNA:DNA duplex confirmed the 3′-endo conformation of an LNA monomer. Recognition of double-stranded DNA has also been demonstrated suggesting strand invasion by LNA. Studies of mismatched sequences show that LNAs obey the Watson-Crick base pairing rules with generally improved selectivity compared to the corresponding unmodified reference strands.

Novel types of LNA-oligomeric compounds, as well as the LNAs, are useful in a wide range of diagnostic and therapeutic applications. Among these are antisense applications, PCR applications, strand-displacement oligomers, substrates for nucleic acid polymerases and generally as nucleotide based drugs.

Potent and nontoxic antisense oligonucleotides containing LNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638.) The authors have demonstrated that LNAs confer several desired properties to antisense agents. LNA/DNA copolymers were not degraded readily in blood serum and cell extracts. LNA/DNA copolymers exhibited potent antisense activity in assay systems as disparate as G-protein-coupled receptor signaling in living rat brain and detection of reporter genes in Escherichia coli. LIPOFECTIN™-mediated efficient delivery of LNA into living human breast cancer cells has also been accomplished.

The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.

The first analogs of LNA, phosphorothioate-LNA and 2′-thio-LNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., PCT International Application WO 98-DK393 19980914). Furthermore, synthesis of 2′-amino-LNA, a novel conformationally restricted high-affinity oligonucleotide analog with a handle has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2′-Amino- and 2′-methylamino-LNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.

Some oligonucleotide mimetics have been prepared to include bicyclic and tricyclic nucleoside analogs having the formulas (amidite monomers shown):

(see Steffens et al., Helv. Chim. Acta, 1997, 80, 2426-2439; Steffens et al., J. Am. Chem. Soc., 1999, 121, 3249-3255; and Renneberg et al., J. Am. Chem. Soc., 2002, 124, 5993-6002). These modified nucleoside analogs have been oligomerized using the phosphoramidite approach and the resulting oligomeric compounds containing tricyclic nucleoside analogs have shown increased thermal stabilities (Tm's) when hybridized to DNA, RNA and itself. Oligomeric compounds containing bicyclic nucleoside analogs have shown thermal stabilities approaching that of DNA duplexes.

Another class of oligonucleotide mimetic is referred to as phosphonomonoester nucleic acid and incorporates a phosphorus group in the backbone. This class of olignucleotide mimetic is reported to have useful physical and biological and pharmacological properties in the areas of inhibiting gene expression (antisense oligonucleotides, ribozymes, sense oligonucleotides and triplex-forming oligonucleotides), as probes for the detection of nucleic acids and as auxiliaries for use in molecular biology.

The general formula (for definitions of Markush variables see: U.S. Pat. Nos. 5,874,553 and 6,127,346 herein incorporated by reference in their entirety) is shown below.

Another oligonucleotide mimetic has been reported wherein the furanosyl ring has been replaced by a cyclobutyl moiety.

Modified Sugars

Oligomeric compounds of the invention may also contain one or more substituted sugar moieties. These oligomeric compounds comprise a sugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly suitable are O((CH₂)_(n)O)_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON((CH₂)_(n)CH₃)₂, where n and m are from 1 to about 10. Some oligonucleotides comprise a sugar substituent group selected from: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. One modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. One modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₃)₂.

Other sugar substituent groups include methoxy (—O—CH₃), aminopropoxy (—OCH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂), —O-allyl (—O—CH₂—CH═CH₂) and fluoro (F). 2′-Sugar substituent groups may be in the arabino (up) position or ribo (down) position. One 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligomeric compound, particularly the 3′ position of the sugar on the 3′ terminal nucleoside or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligomeric compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. No. 4,981,957; U.S. Pat. No. 5,118,800; U.S. Pat. No. 5,319,080; U.S. Pat. No. 5,359,044; U.S. Pat. No. 5,393,878; U.S. Pat. No. 5,446,137; U.S. Pat. No. 5,466,786; U.S. Pat. No. 5,514,785; U.S. Pat. No. 5,519,134; U.S. Pat. No. 5,567,811; U.S. Pat. No. 5,576,427; U.S. Pat. No. 5,591,722; U.S. Pat. No. 5,597,909; U.S. Pat. No. 5,610,300; U.S. Pat. No. 5,627,053; U.S. Pat. No. 5,639,873; U.S. Pat. No. 5,646,265; U.S. Pat. No. 5,658,873; U.S. Pat. No. 5,670,633; U.S. Pat. No. 5,792,747; and U.S. Pat. No. 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

Representative sugar substituent groups include groups of formula I_(a) or II_(a):

wherein:

R_(b) is O, S or NH;

R_(d) is a single bond, O, S or C(═O);

R_(e) is C₁-C₁₀ alkyl, N(R_(k))(R_(m)), N(R_(k))(R_(m)), N═C(R_(p))(R_(q)), N═C(R_(p))(R_(r)) or has formula III_(a);

R_(p) and R_(q) are each independently hydrogen or C₁-C₁₀ alkyl;

R_(r) is —R_(x)—R_(y);

each R_(s), R_(t), R_(u) and R_(v) is, independently, hydrogen, C(O)R_(w), substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugate group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;

or optionally, R_(u) and R_(v), together form a phthalimido moiety with the nitrogen atom to which they are attached;

each R_(w) is, independently, substituted or unsubstituted C₁-C₁₀ alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or aryl;

R_(k) is hydrogen, a nitrogen protecting group or —R_(x)-R_(y);

R_(p) is hydrogen, a nitrogen protecting group or —R_(x)-R_(y);

R_(x) is a bond or a linking moiety;

R_(y) is a chemical functional group, a conjugate group or a solid support medium;

each R_(m) and R_(n) is, independently, H, a nitrogen protecting group, substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, alkynyl; NH₃ ⁺, N(R_(u))(R_(v)), guanidino and acyl where said acyl is an acid amide or an ester;

or R_(m) and R_(n), together, are a nitrogen protecting group, are joined in a ring structure that optionally includes an additional heteroatom selected from N and O or are a chemical functional group;

R_(i) is OR_(z), SR_(z), or N(R_(z))₂;

each R_(z) is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C(═NH)N(H)R_(u), C(═O)N(H)R_(u) or OC(═O)N(H)R_(u);

R_(f), R_(g) and R_(h) comprise a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 heteroatoms wherein said heteroatoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic;

R_(j) is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(R_(k))(R_(m))OR_(k), halo, SR_(k) or CN;

m_(a) is 1 to about 10;

each mb is, independently, 0 or 1;

mc is 0 or an integer from 1 to 10;

md is an integer from 1 to 10;

me is from 0, 1 or 2; and

provided that when mc is 0, md is greater than 1.

Representative substituents groups are disclosed in U.S. patent application Ser. No. 09/130,973, filed Aug. 7, 1998, entitled “Capped 2′-Oxyethoxy Oligonucleotides,” hereby incorporated by reference in its entirety.

Representative cyclic substituent groups are disclosed in U.S. patent application Ser. No. 09/123,108, filed Jul. 27, 1998, entitled “RNA Targeted 2′-Oligomeric compounds that are Conformationally Preorganized,” hereby incorporated by reference in its entirety.

Particular sugar substituent groups include O((CH₂)_(n)O)_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON((CH₂)_(n)CH₃))₂, where n and m are from 1 to about 10.

Representative guanidino substituent groups are disclosed in U.S. patent application Ser. No. 09/349,040, entitled “Functionalized Oligomers,” filed Jul. 7, 1999, hereby incorporated by reference in its entirety.

Representative acetamido substituent groups are disclosed in U.S. Pat. No. 6,147,200 which is hereby incorporated by reference in its entirety.

Representative dimethylaminoethyloxyethyl substituent groups are disclosed in International Patent Application PCT/US99/17895, entitled “2′-O-Dimethylaminoethyloxyethyl-Oligomeric compounds”, filed Aug. 6, 1999, hereby incorporated by reference in its entirety.

Synthesis of Chimeric Oligonucleotides:

Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3 “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers.”

(2′-O-Me)-(2′-deoxy)-(2′-O-Me) Chimeric Phosphorothioate Oligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spectrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

(2′-O-(2-Methoxyethyl))-(2′-deoxy)-(2′-O-(Methoxyethyl)) Chimeric Phosphorothioate Oligonucleotides

(2′-O-(2-methoxyethyl))-(2′-deoxy)-(−2′-O-(methoxyethyl)) chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites.

(2′-O-(2-Methoxyethyl)Phosphodiester)-(2′-deoxy Phosphorothioate)-(2′-O-(2-Methoxyethyl) Phosphodiester) Chimeric Oligonucleotides

(2′-O-(2-methoxyethyl phosphodiester)-(2′-deoxy phosphorothioate)-(2′-O-(methoxyethyl) phosphodiester) chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1, 1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

The terms used to describe the conformational geometry of homoduplex nucleic acids are “A Form” for RNA and “B Form” for DNA. The respective conformational geometry for RNA and DNA duplexes was determined from X-ray diffraction analysis of nucleic acid fibers (Arnott and Hukins, Biochem. Biophys. Res. Comm., 1970, 47, 1504.) In general, RNA:RNA duplexes are more stable and have higher melting temperatures (Tm's) than DNA:DNA duplexes (Sanger et al., Principles of Nucleic Acid Structure, 1984, Springer-Verlag; New York, N.Y.; Lesnik et al., Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res., 1997, 25, 2627-2634). The increased stability of RNA has been attributed to several structural features, most notably the improved base stacking interactions that result from an A-form geometry (Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). The presence of the 2′ hydroxyl in RNA biases the sugar toward a C3′ endo pucker, i.e., also designated as Northern pucker, which causes the duplex to favor the A-form geometry. In addition, the 2′ hydroxyl groups of RNA can form a network of water mediated hydrogen bonds that help stabilize the RNA duplex (Egli et al., Biochemistry, 1996, 35, 8489-8494). On the other hand, deoxy nucleic acids prefer a C2′ endo sugar pucker, i.e., also known as Southern pucker, which is thought to impart a less stable B-form geometry (Sanger, W. (1984) Principles of Nucleic Acid Structure, Springer-Verlag, New York, N.Y.). As used herein, B-form geometry is inclusive of both C2′-endo pucker and O4′-endo pucker. This is consistent with Berger, et. al., Nucleic Acids Research, 1998, 26, 2473-2480, who pointed out that in considering the furanose conformations which give rise to B-form duplexes consideration should also be given to a O4′-endo pucker contribution.

DNA:RNA hybrid duplexes, however, are usually less stable than pure RNA:RNA duplexes, and depending on their sequence may be either more or less stable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). The structure of a hybrid duplex is intermediate between A- and B-form geometries, which may result in poor stacking interactions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306; Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al., Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996, 264, 521-533). The stability of the duplex formed between a target RNA and a synthetic sequence is central to therapies such as, but not limited to, antisense mechanisms, including RNase H-mediated and RNA interference mechanisms, as these mechanisms involved the hybridization of a synthetic sequence strand to an RNA target strand. In the case of RNase H, effective inhibition of the mRNA requires that the antisense sequence achieve at least a threshold of hybridization.

One routinely used method of modifying the sugar puckering is the substitution of the sugar at the 2′-position with a substituent group that influences the sugar geometry. The influence on ring conformation is dependent on the nature of the substituent at the 2′-position. A number of different substituents have been studied to determine their sugar puckering effect. For example, 2′-halogens have been studied showing that the 2′-fluoro derivative exhibits the largest population (65%) of the C3′-endo form, and the 2′-iodo exhibits the lowest population (7%). The populations of adenosine (2′-OH) versus deoxyadenosine (2′-H) are 36% and 19%, respectively. Furthermore, the effect of the 2′-fluoro group of adenosine dimers (2′-deoxy-2′-fluoroadenosine-2′-deoxy-2′-fluoro-adenosine) is also correlated to the stabilization of the stacked conformation.

As expected, the relative duplex stability can be enhanced by replacement of 2′-OH groups with 2′-F groups thereby increasing the C3′-endo population. It is assumed that the highly polar nature of the 2′-F bond and the extreme preference for C3′-endo puckering may stabilize the stacked conformation in an A-form duplex. Data from UV hypochromicity, circular dichroism, and ¹H NMR also indicate that the degree of stacking decreases as the electronegativity of the halo substituent decreases. Furthermore, steric bulk at the 2′-position of the sugar moiety is better accommodated in an A-form duplex than a B-form duplex. Thus, a 2′-substituent on the 3′-terminus of a dinucleoside monophosphate is thought to exert a number of effects on the stacking conformation: steric repulsion, furanose puckering preference, electrostatic repulsion, hydrophobic attraction, and hydrogen bonding capabilities. These substituent effects are thought to be determined by the molecular size, electronegativity, and hydrophobicity of the substituent. Melting temperatures of complementary strands is also increased with the 2′-substituted adenosine diphosphates. It is not clear whether the 3′-endo preference of the conformation or the presence of the substituent is responsible for the increased binding. However, greater overlap of adjacent bases (stacking) can be achieved with the 3′-endo conformation.

Nucleoside conformation is influenced by various factors including substitution at the 2′, 3′ or 4′-positions of the pentofuranosyl sugar. Electronegative substituents generally prefer the axial positions, while sterically demanding substituents generally prefer the equatorial positions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984, Springer-Verlag.) Modification of the 2′ position to favor the 3′-endo conformation can be achieved while maintaining the 2′-OH as a recognition element, as illustrated in FIG. 2, below (Gallo et al., Tetrahedron (2001), 57, 5707-5713. Harry-O'kuru et al., J. Org. Chem., (1997), 62(6), 1754-1759 and Tang et al., J. Org. Chem. (1999), 64, 747-754.) Alternatively, preference for the 3′-endo conformation can be achieved by deletion of the 2′-OH as exemplified by 2′deoxy-2′F-nucleosides (Kawasaki et al., J. Med. Chem. (1993), 36, 831-841), which adopts the 3′-endo conformation positioning the electronegative fluorine atom in the axial position. Other modifications of the ribose ring, for example substitution at the 4′-position to give 4′-F modified nucleosides (Guillerm et al., Bioorganic and Medicinal Chemistry Letters (1995), 5, 1455-1460 and Owen et al., J. Org. Chem. (1976), 41, 3010-3017), or for example modification to yield methanocarba nucleoside analogs (Jacobson et al., J. Med. Chem. Lett. (2000), 43, 2196-2203 and Lee et al., Bioorganic and Medicinal Chemistry Letters (2001), 11, 1333-1337) also induce preference for the 3′-endo conformation.

In one aspect of the present invention oligomeric compounds include nucleosides synthetically modified to induce a 3′-endo sugar conformation. A nucleoside can incorporate synthetic modifications of the heterocyclic base, the sugar moiety or both to induce a desired 3′-endo sugar conformation. These modified nucleosides are used to mimic RNA-like nucleosides so that particular properties of an oligomeric compound can be enhanced while maintaining the desirable 3′-endo conformational geometry (see Scheme 1). There is an apparent preference for an RNA type duplex (A form helix, predominantly 3′-endo) as a requirement (e.g. trigger) of RNA interference which is supported in part by the fact that duplexes composed of 2′-deoxy-2′-F-nucleosides appears efficient in triggering RNAi response in the C. elegans system. Properties that are enhanced by using more stable 3′-endo nucleosides include but aren't limited to modulation of pharmacokinetic properties through modification of protein binding, protein off-rate, absorption and clearance; modulation of nuclease stability as well as chemical stability; modulation of the binding affinity and specificity of the oligomer (affinity and specificity for enzymes as well as for complementary sequences); and increasing efficacy of RNA cleavage. The present invention provides oligomeric compounds designed to act as triggers of RNAi having one or more nucleosides modified in such a way as to favor a C3′-endo type conformation.

Along similar lines, oligomeric triggers of RNAi response might be composed of one or more nucleosides modified in such a way that conformation is locked into a C3′-endo type conformation, i.e. Locked Nucleic Acid (LNA, Singh et al, Chem. Commun. (1998), 4, 455-456), and ethylene bridged Nucleic Acids (ENA, Morita et al, Bioorganic & Medicinal Chemistry Letters (2002), 12, 73-76.) Examples of modified nucleosides amenable to the present invention are shown below. These examples are meant to be representative and not exhaustive.

Oligomeric compounds may also include nucleobase (often referred to in the art simply as “base” or “heterocyclic base moiety”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases also referred herein as heterocyclic base moieties include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Some nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

In one aspect of the present invention oligomeric compounds are prepared having polycyclic heterocyclic compounds in place of one or more heterocyclic base moieties. A number of tricyclic heterocyclic compounds have been previously reported. These compounds are routinely used in antisense applications to increase the binding properties of the modified strand to a target strand. The most studied modifications are targeted to guanosines hence they have been termed G-clamps or cytidine analogs. Many of these polycyclic heterocyclic compounds have the general formula:

Representative cytosine analogs that make 3 hydrogen bonds with a guanosine in a second strand include 1,3-diazaphenoxazine-2-one (R₁₀₌O, R₁₁-R₁₄═H) (Kurchavov, et al, Nucleosides and Nucleotides, 1997, 16, 1837-1846), 1,3-diazaphenothiazine-2-one (R₁₀═S, R₁₁-R₁₄═H), (Lin, K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117, 3873-3874) and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (R₁₀═O, R₁₁-R₁₄═F) (Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998, 39, 8385-8388). When incorporated into oligonucleotides, these base modifications were shown to hybridize with complementary guanine and the latter was also shown to hybridize with adenine and to enhance helical thermal stability by extended stacking interactions (also see U.S. Patent Application Publication 20030207804 and U.S. Patent Application Publication 20030175906, both of which are incorporated herein by reference in their entirety).

Helix-stabilizing properties have been observed when a cytosine analog/substitute has an aminoethoxy moiety attached to the rigid 1,3-diazaphenoxazine-2-one scaffold (R₁₀₌O, R₁₁═—O—(CH₂)₂—NH₂, R₁₂₋₁₄═H) (Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532). Binding studies demonstrated that a single incorporation could enhance the binding affinity of a model oligonucleotide to its complementary target DNA or RNA with a ΔT_(m) of up to 18° relative to 5-methyl cytosine (dC5^(me)), which is the highest known affinity enhancement for a single modification. On the other hand, the gain in helical stability does not compromise the specificity of the oligonucleotides. The T_(m) data indicate an even greater discrimination between the perfect match and mismatched sequences compared to dC5^(me). It was suggested that the tethered amino group serves as an additional hydrogen bond donor to interact with the Hoogsteen face, namely the O6, of a complementary guanine thereby forming 4 hydrogen bonds. This means that the increased affinity of G-clamp is mediated by the combination of extended base stacking and additional specific hydrogen bonding.

Tricyclic heterocyclic compounds and methods of using them that are amenable to the present invention are disclosed in U.S. Pat. No. 6,028,183, and U.S. Pat. No. 6,007,992, the contents of both are incorporated herein in their entirety.

The enhanced binding affinity of the phenoxazine derivatives together with their sequence specificity makes them valuable nucleobase analogs for the development of more potent antisense-based drugs. In fact, promising data have been derived from in vitro experiments demonstrating that heptanucleotides containing phenoxazine substitutions can activate RNaseH, enhance cellular uptake and exhibit an increased antisense activity (Lin, K-Y; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532). The activity enhancement was even more pronounced in case of G-clamp, as a single substitution was shown to significantly improve the in vitro potency of a 20mer 2′-deoxyphosphorothioate oligonucleotides (Flanagan, W. M.; Wolf, J. J.; Olson, P.; Grant, D.; Lin, K.-Y.; Wagner, R. W.; Matteucci, M. Proc. Natl. Acad. Sci. USA, 1999, 96, 3513-3518).

Modified polycyclic heterocyclic compounds useful as heterocyclic bases are disclosed in but not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. No. 4,845,205; U.S. Pat. No. 5,130,302; U.S. Pat. No. 5,134,066; U.S. Pat. No. 5,175,273; U.S. Pat. No. 5,367,066; U.S. Pat. No. 5,432,272; U.S. Pat. No. 5,434,257; U.S. Pat. No. 5,457,187; U.S. Pat. No. 5,459,255; U.S. Pat. No. 5,484,908; U.S. Pat. No. 5,502,177; U.S. Pat. No. 5,525,711; U.S. Pat. No. 5,552,540; U.S. Pat. No. 5,587,469; U.S. Pat. No. 5,594,121, U.S. Pat. No. 5,596,091; U.S. Pat. No. 5,614,617; U.S. Pat. No. 5,645,985; U.S. Pat. No. 5,646,269; U.S. Pat. No. 5,750,692; U.S. Pat. No. 5,830,653; U.S. Pat. No. 5,763,588; U.S. Pat. No. 6,005,096; and U.S. Pat. No. 5,681,941, and U.S. Patent Application Publication 20030158403, each of which is incorporated herein by reference in its entirety.

One substitution that can be appended to the oligomeric compounds of the invention involves the linkage of one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting oligomeric compounds. In one embodiment such modified oligomeric compounds are prepared by covalently attaching conjugate groups to functional groups such as hydroxyl or amino groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates groups include cholesterols, carbohydrates, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

The oligomeric compounds of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.

Representative U.S. patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. No. 4,828,979; U.S. Pat. No. 4,948,882; U.S. Pat. No. 5,218,105; U.S. Pat. No. 5,525,465; U.S. Pat. No. 5,541,313; U.S. Pat. No. 5,545,730; U.S. Pat. No. 5,552,538; U.S. Pat. No. 5,578,717, U.S. Pat. No. 5,580,731; U.S. Pat. No. 5,580,731; U.S. Pat. No. 5,591,584; U.S. Pat. No. 5,109,124; U.S. Pat. No. 5,118,802; U.S. Pat. No. 5,138,045; U.S. Pat. No. 5,414,077; U.S. Pat. No. 5,486,603; U.S. Pat. No. 5,512,439; U.S. Pat. No. 5,578,718; U.S. Pat. No. 5,608,046; U.S. Pat. No. 4,587,044; U.S. Pat. No. 4,605,735; U.S. Pat. No. 4,667,025; U.S. Pat. No. 4,762,779; U.S. Pat. No. 4,789,737; U.S. Pat. No. 4,824,941; U.S. Pat. No. 4,835,263; U.S. Pat. No. 4,876,335; U.S. Pat. No. 4,904,582; U.S. Pat. No. 4,958,013; U.S. Pat. No. 5,082,830; U.S. Pat. No. 5,112,963; U.S. Pat. No. 5,214,136; U.S. Pat. No. 5,082,830; U.S. Pat. No. 5,112,963; U.S. Pat. No. 5,214,136; U.S. Pat. No. 5,245,022; U.S. Pat. No. 5,254,469; U.S. Pat. No. 5,258,506; U.S. Pat. No. 5,262,536; U.S. Pat. No. 5,272,250; U.S. Pat. No. 5,292,873; U.S. Pat. No. 5,317,098; U.S. Pat. No. 5,371,241, U.S. Pat. No. 5,391,723; U.S. Pat. No. 5,416,203, U.S. Pat. No. 5,451,463; U.S. Pat. No. 5,510,475; U.S. Pat. No. 5,512,667; U.S. Pat. No. 5,514,785; U.S. Pat. No. 5,565,552; U.S. Pat. No. 5,567,810; U.S. Pat. No. 5,574,142; U.S. Pat. No. 5,585,481; U.S. Pat. No. 5,587,371; U.S. Pat. No. 5,595,726; U.S. Pat. No. 5,597,696; U.S. Pat. No. 5,599,923; U.S. Pat. No. 5,599,928 and U.S. Pat. No. 5,688,941, each of which is herein incorporated by reference.

Oligomeric compounds used in the compositions of the present invention can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of oligomeric compounds to enhance properties such as for example nuclease stability. Included in stabilizing groups are cap structures. By “cap structure or terminal cap moiety” is meant chemical modifications, which have been incorporated at either terminus of oligonucleotides (see for example Wincott et al., WO 97/26270, incorporated by reference herein). These terminal modifications protect the oligomeric compounds having terminal nucleic acid molecules from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5′-terminus (5′-cap) or at the 3′-terminus (3′-cap) or can be present on both termini. For double-stranded oligomeric compounds, the cap may be present at either or both termini of either strand. In non-limiting examples, the 5′-cap includes inverted abasic residue (moiety), 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl riucleotide, 3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety; 1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety (see Wincott et al., International PCT publication No. WO 97/26270, incorporated by reference herein).

Particularly preferred 3′-cap structures of the present invention include, for example 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5′-mercapto moieties (for more details see Beaucage and Tyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).

Further 3′ and 5′-stabilizing groups that can be used to cap one or both ends of an oligomeric compound to impart nuclease stability include those disclosed in WO 03/004602 published on Jan. 16, 2003.

It is not necessary for all positions in an oligomeric compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single oligomeric compound or even at a single monomeric subunit such as a nucleoside within a oligomeric compound. The present invention also includes oligomeric compounds which are chimeric oligomeric compounds. “Chimeric” oligomeric compounds or “chimeras,” in the context of this invention, are oligomeric compounds that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a nucleic acid based oligomer.

Chimeric oligomeric compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligomeric compound may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, an oligomeric compound may be designed to comprise a region that serves as a substrate for RNase H. RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H by an oligomeric compound having a cleavage region, therefore, results in cleavage of the RNA target, thereby enhancing the efficiency of the oligomeric compound. Consequently, comparable results can often be obtained with shorter oligomeric compounds having substrate regions when chimeras are used, compared to for example phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

Chimeric oligomeric compounds of the invention may be formed as composite structures of two or more oligonucleotides, oligonucleotide mimics, oligonucleotide analogs, oligonucleosides and/or oligonucleotide mimetics as described above. Such oligomeric compounds have also been referred to in the art as hybrids, hemimers, gapmers or inverted gapmers. Representative U.S. patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. No. 5,013,830; U.S. Pat. No. 5,149,797; U.S. Pat. No. 5,220,007; U.S. Pat. No. 5,256,775; U.S. Pat. No. 5,366,878; U.S. Pat. No. 5,403,711; U.S. Pat. No. 5,491,133; U.S. Pat. No. 5,565,350; U.S. Pat. No. 5,623,065; U.S. Pat. No. 5,652,355; U.S. Pat. No. 5,652,356; and U.S. Pat. No. 5,700,922, each of which is herein incorporated by reference in its entirety.

The conformation of modified nucleosides and their oligomers can be estimated by various methods such as molecular dynamics calculations, nuclear magnetic resonance spectroscopy and CD measurements. Hence, modifications predicted to induce RNA-like conformations (A-form duplex geometry in an oligomeric context), are useful in the oligomeric compounds of the present invention. The synthesis of modified nucleosides amenable to the present invention are known in the art (see for example, Chemistry of Nucleosides and Nucleotides Vol 1-3, ed. Leroy B. Townsend, 1988, Plenum Press.)

In one aspect, the present invention is directed to oligomeric compounds that are designed to have enhanced properties compared to native RNA. One method to design optimized or enhanced oligomeric compounds involves each nucleoside of the selected sequence being scrutinized for possible enhancing modifications. One modification would be the replacement of one or more RNA nucleosides with nucleosides that have the same 3′-endo conformational geometry. Such modifications can enhance chemical and nuclease stability relative to native RNA while at the same time being much cheaper and easier to synthesize and/or incorporate into an oligonucleotide. The sequence can be further divided into regions and the nucleosides of each region evaluated for enhancing modifications that can be the result of a chimeric configuration. Consideration is also given to the 5′ and 3′-termini as there are often advantageous modifications that can be made to one or more of the terminal nucleosides. The oligomeric compounds of the present invention may include at least one 5′-modified phosphate group on a single strand or on at least one 5′-position of a double-stranded sequence or sequences. Other modifications considered are internucleoside linkages, conjugate groups, substitute sugars or bases, substitution of one or more nucleosides with nucleoside mimetics and any other modification that can enhance the desired property of the oligomeric compound.

One synthetic 2′-modification that imparts increased nuclease resistance and a very high binding affinity to nucleotides is the 2-methoxyethoxy (2′-MOE, 2′-OCH₂CH₂OCH₃) side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). One of the immediate advantages of the 2′-MOE substitution is the improvement in binding affinity, which is greater than many similar 2′ modifications such as O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2′-O-methoxyethyl substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926). Relative to DNA, the oligonucleotides having the 2′-MOE modification displayed improved RNA affinity and higher nuclease resistance. Chimeric oligonucleotides having 2′-MOE substituents in the wing nucleosides and an internal region of deoxy-phosphorothioate nucleotides (also termed a gapped oligonucleotide or gapmer) have shown effective reduction in the growth of tumors in animal models at low doses. 2′-MOE substituted oligonucleotides have also shown outstanding promise as antisense agents in several disease states. One such MOE substituted oligonucleotide is presently being investigated in clinical trials for the treatment of CMV retinitis.

Unless otherwise defined herein, alkyl means C₁-C₁₂, C₁-C₈, or C₁-C₆, straight or (where possible) branched chain aliphatic hydrocarbyl.

Unless otherwise defined herein, heteroalkyl means C₁-C₁₂, C₁-C₈, or C₁-C₆, straight or (where possible) branched chain aliphatic hydrocarbyl containing at least one, or about 1 to about 3 hetero atoms in the chain, including the terminal portion of the chain. Suitable heteroatoms include N, O and S.

Unless otherwise defined herein, cycloalkyl means C₃-C₁₂, C₃-C₈, or C₃-C₆, aliphatic hydrocarbyl ring.

Unless otherwise defined herein, alkenyl means C₂-C₁₂, C₂-C₈, or C₂-C₆ alkenyl, which may be straight or (where possible) branched hydrocarbyl moiety, which contains at least one carbon-carbon double bond.

Unless otherwise defined herein, alkynyl means C₂-C₁₂, C₂-C₈, or C₂-C₆ alkynyl, which may be straight or (where possible) branched hydrocarbyl moiety, which contains at least one carbon-carbon triple bond.

Unless otherwise defined herein, heterocycloalkyl means a ring moiety containing at least three ring members, at least one of which is carbon, and of which 1, 2 or three ring members are other than carbon. The number of carbon atoms can vary from 1 to about 12, from 1 to about 6, and the total number of ring members varies from three to about 15, or from about 3 to about 8. Suitable ring heteroatoms are N, O and S. Suitable heterocycloalkyl groups include, but are not limited to, morpholino, thiomorpholino, piperidinyl, piperazinyl, homopiperidinyl, homopiperazinyl, homomorpholino, homothiomorpholino, pyrrolodinyl, tetrahydrooxazolyl, tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydroisoxazolyl, tetrahydropyrrazolyl, furanyl, pyranyl, and tetrahydroisothiazolyl.

Unless otherwise defined herein, aryl means any hydrocarbon ring structure containing at least one aryl ring. Suitable aryl rings have about 6 to about 20 ring carbons. Especially suitable aryl rings include phenyl, napthyl, anthracenyl, and phenanthrenyl.

Unless otherwise defined herein, hetaryl means a ring moiety containing at least one fully unsaturated ring, the ring consisting of carbon and non-carbon atoms. The ring system can contain about 1 to about 4 rings. The number of carbon atoms can vary from 1 to about 12, from 1 to about 6, and the total number of ring members varies from three to about 15, or from about 3 to about 8. Suitable ring heteroatoms are N, O and S. Suitable hetaryl moieties include, but are not limited to, pyrazolyl, thiophenyl, pyridyl, imidazolyl, tetrazolyl, pyridyl, pyrimidinyl, purinyl, quinazolinyl, quinoxalinyl, benzimidazolyl, benzothiophenyl, etc.

Unless otherwise defined herein, where a moiety is defined as a compound moiety, such as hetarylalkyl (hetaryl and alkyl), aralkyl (aryl and alkyl), etc., each of the sub-moieties is as defined herein.

Unless otherwise defined herein, an electron withdrawing group is a group, such as the cyano or isocyanato group that draws electronic charge away from the carbon to which it is attached. Other electron withdrawing groups of note include those whose electronegativities exceed that of carbon, for example halogen, nitro, or phenyl substituted in the ortho- or para-position with one or more cyano, isothiocyanato, nitro or halo groups.

Unless otherwise defined herein, the terms halogen and halo have their ordinary meanings. Suitable halo (halogen) substituents are Cl, Br, and I.

The aforementioned optional substituents are, unless otherwise herein defined, suitable substituents depending upon desired properties. Included are halogens (Cl, Br, I), alkyl, alkenyl, and alkynyl moieties, NO₂, NH₃ (substituted and unsubstituted), acid moieties (e.g. —CO₂H, —OSO₃H₂, etc.), heterocycloalkyl moieties, hetaryl moieties, aryl moieties, etc.

In all the preceding formulae, the squiggle (˜) indicates a bond to an oxygen or sulfur of the 5′-phosphate.

Phosphate protecting groups include those described in U.S. Pat. No. 5,760,209, U.S. Pat. No. 5,614,621, U.S. Pat. No. 6,051,699, U.S. Pat. No. 6,020,475, U.S. Pat. No. 6,326,478, U.S. Pat. No. 6,169,177, U.S. Pat. No. 6,121,437, U.S. Pat. No. 6,465,628 each of which is expressly incorporated herein by reference in its entirety.

Screening methods for the identification of effective modulators of small non-coding RNAs are also comprehended by the instant invention and comprise the steps of contacting a small non-coding RNA, or portion thereof, with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the levels, expression or alter the function of the small non-coding RNA. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the levels, expression or altering the function of the small non-coding RNA, the modulator may then be employed in further investigative studies, or for use as a target validation, research, diagnostic, or therapeutic agent in accordance with the present invention.

Screening methods for the identification of small non-coding RNA mimics are also within the scope of the invention. Screening for small non-coding RNA modulators or mimics can also be performed in vitro, ex vivo, or in vivo by contacting samples, tissues, cells or organisms with candidate modulators or mimics and selecting for one or more candidate modulators which show modulatory effects.

Design and Screening of Duplexed Oligomeric Compounds:

In screening and target validation studies, oligomeric compounds of the invention can be used in combination with their respective complementary strand oligomeric compound to form stabilized double-stranded (duplexed) oligonucleotides. In accordance with the present invention, a series of duplexes comprising the oligomeric compounds of the present invention and their complements can be designed to target a small non-coding RNA. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in some embodiments, both strands of the duplex would be complementary over the central nucleobases, each having overhangs at one or both termini, as described supra.

In some embodiments, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO:2181) may be prepared with blunt ends (no single stranded overhang) as shown:

In other embodiments, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG, having a two-nucleobase overhang of deoxythymidine (dT) and its complement sense strand may be prepared with overhangs as shown:

RNA strands of the duplex can be synthesized by methods disclosed herein or purchased from Dharmacon Research Inc., (Lafayette, Colo.).

For use in drug discovery, oligomeric compounds of the present invention are used to elucidate relationships that exist between small non-coding RNAs, genes or proteins and a disease state, phenotype, or condition. These methods include detecting or modulating a target comprising contacting a sample, tissue, cell, or organism with the oligomeric compounds and compositions of the present invention, measuring the levels of the target and/or the levels of downstream gene products including mRNA or proteins encoded thereby, a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to an untreated sample, a positive control or a negative control. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a disease.

The oligomeric compounds and compositions of the present invention can additionally be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. Such uses allows for those of ordinary skill to elucidate the function of particular non-coding or coding nucleic acids or to distinguish between functions of various members of a biological pathway.

For use in kits and diagnostics, the oligomeric compounds and compositions of the present invention, either alone or in combination with other compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of non-coding or coding nucleic acids expressed within cells and tissues.

As one non-limiting example, expression patterns within cells or tissues treated with one or more oligomeric compounds or compositions of the invention are compared to control cells or tissues not treated with the compounds or compositions and the patterns produced are analyzed for differential levels of nucleic acid expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds that affect expression patterns.

Cell Culture and Oligonucleotide Treatment:

The effects of oligomeric compounds on target nucleic acid expression or function can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or real-time RT-PCR. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is present in the cell type chosen.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 is obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. For Northern blotting or other analyses, cells harvested when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #353872) at a density of 7000 cells/well for use in real-time RT-PCR analysis.

A549 Cells:

The human lung carcinoma cell line A549 is obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

HMECs:

Normal human mammary epithelial cells (HMECs) are obtained from American Type Culture Collection (Manassus, Va.). HMECs are routinely cultured in DMEM high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reach approximately 90% confluence. HMECs are plated in 24-well plates (Falcon-Primaria #353047, BD Biosciences, Bedford, Mass.) at a density of 50,000-60,000 cells per well, and allowed to attach overnight prior to treatment with oligomeric compounds. HMECs are plated in 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a density of approximately 10,000 cells per well and allowed to attach overnight prior to treatment with oligomeric compounds.

MCF7 Cells:

The breast carcinoma cell line MCF7 is obtained from American Type Culture Collection (Manassus, Va.). MCF7 cells are routinely cultured in DMEM high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reach approximately 90% confluence. MCF7 cells are plated in 24-well plates (Falcon-Primaria #353047, BD Biosciences, Bedford, Mass.) at a density of approximately 140,000 cells per well, and allowed to attach overnight prior to treatment with oligomeric compounds. MCF7 cells are plated in 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a density of approximately 20,000 cells per well and allowed to attach overnight prior to treatment with oligomeric compounds.

T47D Cells:

The breast carcinoma cell line T47D is obtained from American Type Culture Collection (Manassus, Va.). T47D cells are deficient in expression of the tumor suppressor gene p53. T47D cells are cultured in DMEM high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reach approximately 90% confluence. T47D cells are plated in 24-well plates (Falcon-Primaria #353047, BD Biosciences, Bedford, Mass.) at a density of approximately 170,000 cells per well, and allowed to attach overnight prior to treatment with oligomeric compounds. T47D cells are plated in 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a density of approximately 20,000 cells per well and allowed to attach overnight prior to treatment with oligomeric compounds.

BJ Cells:

The normal human foreskin fibroblast BJ cell line was obtained from American Type Culture Collection (Manassus, Va.). BJ cells were routinely cultured in MEM high glucose with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate and supplemented with 10% fetal bovine serum, 0.1 mM non-essential amino acids and 1.0 mM sodium pyruvate (all media and supplements from Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached approximately 80% confluence. Cells were plated on collagen-coated 24-well plates (Falcon-Primaria #3047, BD Biosciences, Bedford, Mass.) at approximately 50,000 cells per well, and allowed to attach to wells overnight.

B16-F10 Cells:

The mouse melanoma cell line B16-F10 was obtained from American Type Culture Collection (Manassas, Va.). B16-F10 cells were routinely cultured in DMEM high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached approximately 80% confluence. Cells were seeded into collagen-coated 24-well plates (Falcon-Primaria #3047, BD Biosciences, Bedford, Mass.) at approximately 50,000 cells per well and allowed to attach overnight.

HUVECs:

Human vascular endothelial cells (HUVECs) are obtained from American Type Culture Collection (Manassus, Va.). HUVECs are routinely cultured in EBM (Clonetics Corporation, Walkersville, Md.) supplemented with SingleQuots supplements (Clonetics Corporation, Walkersville, Md.). Cells are routinely passaged by trypsinization and dilution when they reach approximately 90% confluence and are maintained for up to 15 passages. HUVECs are plated at approximately 3000 cells/well in 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) and treated with oligomeric compounds one day later.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) cells are obtained from the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) are obtained from the Clonetics Corporation (Walkersville, Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

293T Cells:

The human 293T cell line is obtained from American Type Culture Collection (Manassas, Va.). 293T cells are a highly transfectable cell line constitutively expressing the simian virus 40 (SV40) large T antigen. 293T cells were maintained in Dulbeccos' Modified Medium (DMEM) (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum and antibiotics (Life Technologies).

HepG2 Cells:

The human hepatoblastoma cell line HepG2 is obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). HepG2 cells are routinely cultured in Eagle's MEM supplemented with 10% fetal bovine serum, 1 mM non-essential amino acids, and 1 mM sodium pyruvate (medium and all supplements from Invitrogen Life Technologies, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reach approximately 90% confluence. For treatment with oligomeric compounds, cells are seeded into 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a density of approximately 7000 cells/well prior to treatment with oligomeric compounds. For the caspase assay, cells are seeded into collagen coated 96-well plates (BIOCOAT cellware, Collagen type I, B-D #354407/356407, Becton Dickinson, Bedford, Mass.) at a density of 7500 cells/well.

Preadipocytes:

Human preadipocytes are obtained from Zen-Bio, Inc. (Research Triangle Park, N.C.). Preadipocytes were routinely maintained in Preadipocyte Medium (ZenBio, Inc., Research Triangle Park, N.C.) supplemented with antibiotics as recommended by the supplier. Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were routinely maintained for up to 5 passages as recommended by the supplier. To induce differentiation of preadipocytes, cells are then incubated with differentiation media consisting of Preadipocyte Medium further supplemented with 2% more fetal bovine serum (final total of 12%), amino acids, 100 nM insulin, 0.5 mM IBMX, 1 μM dexamethasone and 1 μM BRL49653. Cells are left in differentiation media for 3-5 days and then re-fed with adipocyte media consisting of Preadipocyte Medium supplemented with 33 μM biotin, 17 μM pantothenate, 100 nM insulin and 1 μM dexamethasone. Cells differentiate within one week. At this point cells are ready for treatment with the oligomeric compounds of the invention. One day prior to transfection, 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) are seeded with approximately 3000 cells/well prior to treatment with oligomeric compounds.

Differentiated Adipocytes:

Human adipocytes are obtained from Zen-Bio, Inc. (Research Triangle Park, N.C.). Adipocytes were routinely maintained in Adipocyte Medium (ZenBio, Inc., Research Triangle Park, N.C.) supplemented with antibiotics as recommended by the supplier. Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were routinely maintained for up to 5 passages as recommended by the supplier.

NT2 Cells:

The NT2 cell line is obtained from the American Type Culture Collection (ATCC; Manassa, Va.). The NT2 cell line, which has the ATCC designation NTERA-2 c1.D1, is a pluripotent human testicular embryonal carcinoma cell line derived by cloning the NTERA-2 cell line. The parental NTERA-2 line was established in 1980 from a nude mouse xenograft of the Tera-2 cell line (ATCC HTB-106). NT2 cells were routinely cultured in DMEM, high glucose (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. For Northern blotting or other analyses, cells harvested when they reached 90% confluence.

HeLa Cells:

The human epitheloid carcinoma cell line HeLa is obtained from the American Tissue Type Culture Collection (Manassas, Va.). HeLa cells were routinely cultured in DMEM, high glucose (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. For Northern blotting or other analyses, cells were harvested when they reached 90% confluence.

For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

Treatment with Antisense Oligomeric Compounds:

In general, when cells reach approximately 80% confluency, they are treated with oligomeric compounds of the invention. Oligomeric compounds are introduced into cells using the cationic lipid transfection reagent LIPOFECTIN™ (Invitrogen Life Technologies, Carlsbad, Calif.). Oligomeric compounds are mixed with LIPOFECTIN™ in OPTI-MEM™ (Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the desired final concentration of oligomeric compound and LIPOFECTIN™. Before adding to cells, the oligomeric compound, LIPOFECTIN™ and OPTI-MEM™ are mixed thoroughly and incubated for approximately 0.5 hrs. The medium is removed from the plates and the plates are tapped on sterile gauze. Each well of a 96-well plate is washed with 150 μl of phosphate-buffered saline or Hank's balanced salt solution. Each well of a 24-well plate is washed with 250 μL of phosphate-buffered saline or Hank's balanced salt solution. The wash buffer in each well is replaced with 100 μL or 250 μL of the oligomeric compound/OPTI-MEM™/LIPOFECTIN™ cocktail for 96-well or 24-well plates, respectively. Untreated control cells receive LIPOFECTIN™ only. The plates are incubated for approximately 4 to 7 hours at 37° C., after which the medium is removed and the plates are tapped on sterile gauze. 100 μl or 1 mL of full growth medium is added to each well of a 96-well plate or a 24-well plate, respectively. Cells are harvested 16-24 hours after oligonucleotide treatment, at which time RNA can be isolated and target reduction measured by real-time RT-PCR, or other phenotypic assays performed. In general, data from treated cells are obtained in triplicate, and results presented as an average of the three trials.

In some embodiments, cells are transiently transfected with oligomeric compounds of the instant invention. In some embodiments, cells are transfected and selected for stable expression of an oligomeric compound of the instant invention.

The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide may be selected from ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted to human Jun-N-terminal kinase-2 (JNK2) or another suitable positive control. Controls are 2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone or having chemical modifications similar to the oligonucleotides being tested. For mouse or rat cells the positive control oligonucleotide may be ISIS 15770 (ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3), a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf The concentration of positive control oligonucleotide that results in 80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) or other suitable control target RNA may then be utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of target expression or function is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. The concentrations of oligonucleotides used herein can range from 10 nM to 300 nM.

Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904), mass spectrometry methods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41) and real-time quantitative RT-PCR (Heid, et al., Genome Res., 1996, 6(10), 986-94).

Analysis of Oligonucleotide Inhibition of a Target Levels or Expression:

Modulation of target levels or expression can be assayed in a variety of ways known in the art. For example, target nucleic acid levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time quantitative RT-PCR (also known as RT-PCR). Real-time quantitative RT-PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Real-time quantitative RT-PCR can be conveniently accomplished using the commercially available ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

RNA Isolation:

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al, (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are routine in the art. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold phosphate-buffered saline (PBS). 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 150 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 140 μL of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.

Real-Time Quantitative PCR Analysis of a Target RNA Levels:

Quantitation of a target RNA levels was accomplished by real-time quantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of RNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after oligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer/probe sets specific to the target gene (or RNA) being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene (or RNA) and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, RNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer/probe sets specific for GAPDH only, target gene (or RNA) only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target RNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer/probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.

PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of DATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene (or RNA) target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.

Probes and primers are designed to hybridize to the target sequence.

Northern Blot Analysis of Target RNA Levels:

Eighteen hours after treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

To detect a target, a target specific primer/probe set is prepared for analysis by PCR. To normalize for variations in loading and transfer efficiency, membranes can be stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data can be normalized to GAPDH levels in untreated controls.

The compounds and compositions of the invention are useful for research and diagnostics, because these compounds and compositions hybridize to nucleic acids or interfere with the normal function of these nucleic acids. Hybridization of the compounds and compositions of the invention with a nucleic acid can be detected by means known in the art. Such means may include conjugation of an enzyme to the compound or composition, radiolabeling or any other suitable detection means. Kits using such detection means for detecting the level of selected proteins in a sample may also be prepared.

The specificity and sensitivity of compounds and compositions can also be harnessed by those of skill in the art for therapeutic uses. Antisense oligomeric compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligomeric compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having a disease or disorder presenting conditions that can be treated, ameliorated, or improved by modulating the expression of a selected small non-coding target nucleic acid is treated by administering the compounds and compositions. For example, in one non-limiting embodiment, the methods comprise the step of administering to or contacting the animal, an effective amount of a modulator or mimic to treat, ameliorate or improve the conditions associated with the disease or disorder. The compounds of the present invention effectively modulate the activity or function of the small non-coding RNA target or inhibit the expression or levels of the small non-coding RNA target. In one embodiment, the activity or expression of the target in an animal is inhibited by about 10%. In another embodiment the activity or expression of a target in an animal is inhibited by about 30%. Further, the activity or expression of a target in an animal is inhibited by 50% or more, by 60% or more, by 70% or more, by 80% or more, by 90% or more, or by 95% or more. In another embodiment, the present invention provides for the use of a compound of the invention in the manufacture of a medicament for the treatment of any and all conditions disclosed herein.

The reduction of target levels may be measured in serum, adipose tissue, liver or any other body fluid, tissue or organ of the animal known to contain the small non-coding RNA or its precursor. Further, the cells contained within the fluids, tissues or organs being analyzed contain a nucleic acid molecule of a downstream target regulated or modulated by the small non-coding RNA target itself.

The oligomeric compounds and compositions of the invention can be utilized in pharmaceutical compositions by adding an effective amount of the compound or composition to a suitable pharmaceutically acceptable diluent or carrier. Use of the oligomeric compounds and methods of the invention may also be useful prophylactically.

The oligomeric compounds and compositions of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative U.S. patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. No. 5,108,921; U.S. Pat. No. 5,354,844; U.S. Pat. No. 5,416,016; U.S. Pat. No. 5,459,127; U.S. Pat. No. 5,521,291; U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,547,932; U.S. Pat. No. 5,583,020; U.S. Pat. No. 5,591,721; U.S. Pat. No. 4,426,330; U.S. Pat. No. 4,534,899; U.S. Pat. No. 5,013,556; U.S. Pat. No. 5,108,921; U.S. Pat. No. 5,213,804; U.S. Pat. No. 5,227,170; U.S. Pat. No. 5,264,221; U.S. Pat. No. 5,356,633; U.S. Pat. No. 5,395,619; U.S. Pat. No. 5,416,016; U.S. Pat. No. 5,417,978; U.S. Pat. No. 5,462,854; U.S. Pat. No. 5,469,854; U.S. Pat. No. 5,512,295; U.S. Pat. No. 5,527,528; U.S. Pat. No. 5,534,259; U.S. Pat. No. 5,543,152; U.S. Pat. No. 5,556,948; U.S. Pat. No. 5,580,575; and U.S. Pat. No. 5,595,756, each of which is herein incorporated by reference.

The oligomeric compounds and compositions of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the oligomeric compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligomeric compounds of the invention can be prepared as SATE ((S-acetyl-2-thioethyl) phosphate) derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al, published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al Larger oligomeric compounds that are processed to supply, as cleavage products, compounds capable of modulating the function or expression of small non-coding RNAs or their downstream targets are also considered prodrugs.

The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds and compositions of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. Suitable examples include, but are not limited to, sodium and postassium salts. For oligonucleotides, examples of pharmaceutically acceptable salts and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

The present invention also includes pharmaceutical compositions and formulations that include the oligomeric compounds and compositions of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

Oligomeric compounds may be formulated for delivery in vivo in an acceptable dosage form, e.g. as parenteral or non-parenteral formulations. Parenteral formulations include intravenous (IV), subcutaneous (SC), intraperitoneal (IP), intravitreal and intramuscular (IM) formulations, as well as formulations for delivery via pulmonary inhalation, intranasal administration, topical administration, etc. Non-parenteral formulations include formulations for delivery via the alimentary canal, e.g. oral administration, rectal administration, intrajejunal instillation, etc. Rectal administration includes administration as an enema or a suppository. Oral administration includes administration as a capsule, a gel capsule, a pill, an elixir, etc.

In some embodiments, an oligomeric compound can be administered to a subject via an oral route of administration. The subject may be an animal or a human (man). An animal subject may be a mammal, such as a mouse, a rat, a dog, a guinea pig, a monkey, a non-human primate, a cat or a pig. Non-human primates include monkeys and chimpanzees. A suitable animal subject may be an experimental animal, such as a mouse, rat, mouse, a rat, a dog, a monkey, a non-human primate, a cat or a pig.

In some embodiments, the subject may be a human. In certain embodiments, the subject may be a human patient. In certain embodiments, the subject may be in need of modulation of expression of one or more genes as discussed in more detail herein. In some particular embodiments, the subject may be in need of inhibition of expression of one or more genes as discussed in more detail herein. In particular embodiments, the subject may be in need of modulation, i.e. inhibition or enhancement, of a nucleic acid target in order to obtain therapeutic indications discussed in more detail herein.

In some embodiments, non-parenteral (e.g. oral) oligomeric compound formulations according to the present invention result in enhanced bioavailability of the compound. In this context, the term “bioavailability” refers to a measurement of that portion of an administered drug which reaches the circulatory system (e.g. blood, especially blood plasma) when a particular mode of administration is used to deliver the drug. Enhanced bioavailability refers to a particular mode of administration's ability to deliver oligonucleotide to the peripheral blood plasma of a subject relative to another mode of administration. For example, when a non-parenteral mode of administration (e.g. an oral mode) is used to introduce the drug into a subject, the bioavailability for that mode of administration may be compared to a different mode of administration, e.g. an IV mode of administration. In some embodiments, the area under a compound's blood plasma concentration curve (AUC₀) after non-parenteral (e.g. oral, rectal, intrajejunal) administration may be divided by the area under the drug's plasma concentration curve after intravenous (i.v.) administration (AUC_(iv)) to provide a dimensionless quotient (relative bioavailability, RB) that represents the fraction of compound absorbed via the non-parenteral route as compared to the IV route. A composition's bioavailability is said to be enhanced in comparison to another composition's bioavailability when the first composition's relative bioavailability (RB₁) is greater than the second composition's relative bioavailability (RB₂).

In general, bioavailability correlates with therapeutic efficacy when a compound's therapeutic efficacy is related to the blood concentration achieved, even if the drug's ultimate site of action is intracellular (van Berge-Henegouwen et al., Gastroenterol., 1977, 73, 300). Bioavailability studies have been used to determine the degree of intestinal absorption of a drug by measuring the change in peripheral blood levels of the drug after an oral dose (DiSanto, Chapter 76 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 1451-1458).

In general, an oral composition's bioavailability is said to be “enhanced” when its relative bioavailability is greater than the bioavailability of a composition substantially consisting of pure oligonucleotide, i.e. oligonucleotide in the absence of a penetration enhancer.

Organ bioavailability refers to the concentration of compound in an organ. Organ bioavailability may be measured in test subjects by a number of means, such as by whole-body radiography. Organ bioavailability may be modified, e.g. enhanced, by one or more modifications to the oligomeric compound, by use of one or more carrier compounds or excipients. In general, an increase in bioavailability will result in an increase in organ bioavailability.

Oral oligomeric compound compositions according to the present invention may comprise one or more “mucosal penetration enhancers,” also known as “absorption enhancers” or simply as “penetration enhancers.” Accordingly, some embodiments of the invention comprise at least one oligomeric compound in combination with at least one penetration enhancer. In general, a penetration enhancer is a substance that facilitates the transport of a drug across mucous membrane(s) associated with the desired mode of administration, e.g. intestinal epithelial membranes. Accordingly it is desirable to select one or more penetration enhancers that facilitate the uptake of one or more oligomeric compounds, without interfering with the activity of the compounds, and in such a manner the compounds can be introduced into the body of an animal without unacceptable side-effects such as toxicity, irritation or allergic response.

Embodiments of the present invention provide compositions comprising one or more pharmaceutically acceptable penetration enhancers, and methods of using such compositions, which result in the improved bioavailability of oligomeric compounds administered via non-parenteral modes of administration. Heretofore, certain penetration enhancers have been used to improve the bioavailability of certain drugs. See Muranishi, Crit. Rev. Ther. Drug Carrier Systems, 1990, 7, 1 and Lee et al., Crit. Rev. Ther. Drug Carrier Systems, 1991, 8, 91. It has been found that the uptake and delivery of oligonucleotides can be greatly improved even when administered by non-parenteral means through the use of a number of different classes of penetration enhancers.

In some embodiments, compositions for non-parenteral administration include one or more modifications from naturally-occurring oligonucleotides (i.e. full-phosphodiester deoxyribosyl or full-phosphodiester ribosyl oligonucleotides). Such modifications may increase binding affinity, nuclease stability, cell or tissue permeability, tissue distribution, or other biological or pharmacokinetic property. Modifications may be made to the base, the linker, or the sugar, in general, as discussed in more detail herein with regards to oligonucleotide chemistry. In some embodiments of the invention, compositions for administration to a subject, and in particular oral compositions for administration to an animal or human subject, will comprise modified oligonucleotides having one or more modifications for enhancing affinity, stability, tissue distribution, or other biological property.

Suitable modified linkers include phosphorothioate linkers. In some embodiments according to the invention, the oligomeric compound has at least one phosphorothioate linker. Phosphorothioate linkers provide nuclease stability as well as plasma protein binding characteristics to the compound. Nuclease stability is useful for increasing the in vivo lifetime of oligomeric compounds, while plasma protein binding decreases the rate of first pass clearance of oligomeric compound via renal excretion. In some embodiments according to the present invention, the oligomeric compound has at least two phosphorothioate linkers. In some embodiments, wherein the oligomeric compound has exactly n nucleosides, the oligomeric compound has from one to n−1 phosphorothioate linkages. In some embodiments, wherein the oligomeric compound has exactly n nucleosides, the oligomeric compound has n−1 phosphorothioate linkages. In other embodiments wherein the oligomeric compound has exactly n nucleoside, and n is even, the oligomeric compound has from 1 to n/2 phosphorothioate linkages, or, when n is odd, from 1 to (n−1)/2 phosphorothioate linkages. In some embodiments, the oligomeric compound has alternating phosphodiester (PO) and phosphorothioate (PS) linkages. In other embodiments, the oligomeric compound has at least one stretch of two or more consecutive PO linkages and at least one stretch of two or more PS linkages. In other embodiments, the oligomeric compound has at least two stretches of PO linkages interrupted by at least one PS linkage.

In some embodiments, at least one of the nucleosides is modified on the ribosyl sugar unit by a modification that imparts nuclease stability, binding affinity or some other beneficial biological property to the sugar. In some cases, the sugar modification includes a 2′-modification, e.g. the 2′-OH of the ribosyl sugar is replaced or substituted. Suitable replacements for 2′-OH include 2′-F and 2′-arabino-F. Suitable substitutions for OH include 2′-O-alkyl, e.g. 2′-O-methyl, and 2′-O-substituted alkyl, e.g. 2′-O-methoxyethyl, 2′-O-aminopropyl, etc. In some embodiments, the oligomeric compound contains at least one 2′-modification. In some embodiments, the oligomeric compound contains at least 22′-modifications. In some embodiments, the oligomeric compound has at least one 2′-modification at each of the termini (i.e. the 3′- and 5′-terminal nucleosides each have the same or different 2′-modifications). In some embodiments, the oligomeric compound has at least two sequential 2′-modifications at each end of the compound. In some embodiments, oligomeric compounds further comprise at least one deoxynucleoside. In particular embodiments, oligomeric compounds comprise a stretch of deoxynucleosides such that the stretch is capable of activating RNase (e.g. RNase H) cleavage of an RNA to which the oligomeric compound is capable of hybridizing. In some embodiments, a stretch of deoxynucleosides capable of activating RNase-mediated cleavage of RNA comprises about 8 to about 16, e.g. about 8 to about 16 consecutive deoxynucleosides. In further embodiments, oligomeric compounds are capable of eliciting cleaveage by dsRNAse enzymes.

Oral compositions for administration of non-parenteral oligomeric compounds and compositions of the present invention may be formulated in various dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The term “alimentary delivery” encompasses e.g. oral, rectal, endoscopic and sublingual/buccal administration. A common requirement for these modes of administration is absorption over some portion or all of the alimentary tract and a need for efficient mucosal penetration of the nucleic acid(s) so administered.

Delivery of a drug via the oral mucosa, as in the case of buccal and sublingual administration, has several desirable features, including, in many instances, a more rapid rise in plasma concentration of the drug than via oral delivery (Harvey, Chapter 35 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, page 711).

Endoscopy may be used for delivery directly to an interior portion of the alimentary tract. For example, endoscopic retrograde cystopancreatography (ERCP) takes advantage of extended gastroscopy and permits selective access to the biliary tract and the pancreatic duct (Hirahata et al., Gan To Kagaku Ryoho, 1992, 19(10 Suppl.), 1591). Pharmaceutical compositions, including liposomal formulations, can be delivered directly into portions of the alimentary canal, such as, e.g., the duodenum (Somogyi et al., Pharm. Res., 1995, 12, 149) or the gastric submucosa (Akamo et al., Japanese J. Cancer Res., 1994, 85, 652) via endoscopic means. Gastric lavage devices (Inoue et al., Artif. Organs, 1997, 21, 28) and percutaneous endoscopic feeding devices (Pennington et al., Ailment Pharmacol Ther., 1995, 9, 471) can also be used for direct alimentary delivery of pharmaceutical compositions.

In some embodiments, oligomeric compound formulations may be administered through the anus into the rectum or lower intestine. Rectal suppositories, retention enemas or rectal catheters can be used for this purpose and may be preferred when patient compliance might otherwise be difficult to achieve (e.g., in pediatric and geriatric applications, or when the patient is vomiting or unconscious). Rectal administration can result in more prompt and higher blood levels than the oral route. (Harvey, Chapter 35 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, page 711). Because about 50% of the drug that is absorbed from the rectum will bypass the liver, administration by this route significantly reduces the potential for first-pass metabolism (Benet et al., Chapter 1 In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996).

Some embodiments of the present invention employ various penetration enhancers in order to effect transport of oligomeric compounds and compositions across mucosal and epithelial membranes. Penetration enhancers may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Penetration enhancers and their uses are described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Accordingly, some embodiments comprise oral oligomeric compound compositions comprising at least one member of the group consisting of surfactants, fatty acids, bile salts, chelating agents, and non-chelating surfactants. Further embodiments comprise oral oligomeric compound comprising at least one fatty acid, e.g. capric or lauric acid, or combinations or salts thereof. Other embodiments comprise methods of enhancing the oral bioavailability of an oligomeric compound, the method comprising co-administering the oligomeric compound and at least one penetration enhancer.

Other excipients that may be added to oral oligomeric compound compositions include surfactants (or “surface-active agents”), which are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligomeric compounds through the alimentary mucosa and other epithelial membranes is enhanced. In addition to bile salts and fatty acids, surfactants include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and perfluorohemical emulsions, such as FC-43 (Takahashi et al., J. Pharm. Phamacol., 1988, 40, 252).

Fatty acids and their derivatives which act as penetration enhancers and may be used in compositions of the present invention include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcamitines, acylcholines and mono- and di-glycerides thereof and/or physiologically acceptable salts thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1; El-Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651).

In some embodiments, oligomeric compound compositions for oral delivery comprise at least two discrete phases, which phases may comprise particles, capsules, gel-capsules, microspheres, etc. Each phase may contain one or more oligomeric compounds, penetration enhancers, surfactants, bioadhesives, effervescent agents, or other adjuvant, excipient or diluent. In some embodiments, one phase comprises at least one oligomeric compound and at least one penetration enhancer. In some embodiments, a first phase comprises at least one oligomeric compound and at least one penetration enhancer, while a second phase comprises at least one penetration enhancer. In some embodiments, a first phase comprises at least one oligomeric compound and at least one penetration enhancer, while a second phase comprises at least one penetration enhancer and substantially no oligomeric compound. In some embodiments, at least one phase is compounded with at least one degradation retardant, such as a coating or a matrix, which delays release of the contents of that phase. In some embodiments, a first phase comprises at least one oligomeric compound, at least one penetration enhancer, while a second phase comprises at least one penetration enhancer and a release-retardant. In particular embodiments, an oral oligomeric compound comprises a first phase comprising particles containing an oligomeric compound and a penetration enhancer, and a second phase comprising particles coated with a release-retarding agent and containing penetration enhancer.

A variety of bile salts also function as penetration enhancers to facilitate the uptake and bioavailability of drugs. The physiological roles of bile include the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et at., eds., McGraw-Hill, New York, N.Y., 1996, pages 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term “bile salt” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (CDCA, sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579).

In some embodiments, penetration enhancers useful in some embodiments of present invention are mixtures of penetration enhancing compounds. One such penetration enhancer is a mixture of UDCA (and/or CDCA) with capric and/or lauric acids or salts thereof e.g. sodium. Such mixtures are useful for enhancing the delivery of biologically active substances across mucosal membranes, in particular intestinal mucosa. Other penetration enhancer mixtures comprise about 5-95% of bile acid or salt(s) UDCA and/or CDCA with 5-95% capric and/or lauric acid. Particular penetration enhancers are mixtures of the sodium salts of UDCA, capric acid and lauric acid in a ratio of about 1:2:2 respectively. Another such penetration enhancer is a mixture of capric and lauric acid (or salts thereof) in a 0.01:1 to 1:0.01 ratio (mole basis). In particular embodiments capric acid and lauric acid are present in molar ratios of e.g. about 0.1:1 to about 1:0.1, in particular about 0.5:1 to about 1:0.5.

Other excipients include chelating agents, i.e. compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligomeric compounds through the alimentary and other mucosa is enhanced. With regard to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315). Chelating agents of the invention include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1; Buur et al., J. Control Ret., 1990, 14, 43).

As used herein, non-chelating non-surfactant penetration enhancers may be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligomeric compounds through the alimentary and other mucosal membranes (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1). This class of penetration enhancers includes, but is not limited to, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621).

Agents that enhance uptake of oligomeric compounds at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), can be used.

Some oral oligomeric compound compositions also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which may be inert (i.e., does not possess biological activity per se) or may be necessary for transport, recognition or pathway activation or mediation, or is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of an oligomeric compound having biological activity by, for example, degrading the biologically active oligomeric compound or promoting its removal from circulation. The coadministration of a oligomeric compound and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of oligomeric compound recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the oligomeric compound for a common receptor. For example, the recovery of a partially phosphorothioate oligomeric compound in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., Antisense Res. Dev., 1995, 5, 115; Takakura et al., Antisense & Nucl Acid Drug Dev., 1996, 6, 177).

A “pharmaceutical carrier” or “excipient” may be a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more oligomeric compounds to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with an oligomeric compound and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, EXPLOTAB); and wetting agents (e.g., sodium lauryl sulphate, etc.).

Oral oligomeric compound compositions may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipuritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the composition of present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The oligomeric compounds and compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations.

Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug that may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment of the present invention. Emulsions and their uses are well known in the art and are described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

Formulations of the present invention include liposomal formulations. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged nucleic acid molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap nucleic acids rather than complex with it. Both cationic and noncationic liposomes have been used to deliver nucleic acids and oligomeric compounds to cells.

Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

The pharmaceutical formulations and compositions of the present invention may also include surfactants. The use of surfactants in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.

Formulations for topical administration include those in which the oligomeric compounds of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).

For topical or other administration, oligomeric compounds and compositions of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, they may be complexed to lipids, in particular to cationic lipids. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999, which is incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Oral formulations are those in which oligomeric compounds of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. A particularly suitable combination is the sodium salt of lauric acid, capric acid and UDCA. Penetration enhancers also include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Compounds and compositions of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Certain oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. Nos. 09/108,673 (filed Jul. 1, 1998), 09/315,298 (filed May 20, 1999) and U.S. Application Publication 20030027780, each of which is incorporated herein by reference in their entirety.

Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositions containing one or more of the compounds and compositions of the invention and one or more other chemotherapeutic agents that function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). When used with the oligomeric compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Combinations of oligomeric compounds and compositions of the invention and other drugs are also within the scope of this invention. Two or more combined compounds such as two oligomeric compounds or one oligomeric compound combined with further compounds may be used together or sequentially.

In another embodiment, compositions of the invention may contain one or more of the compounds and compositions of the invention targeted to a first nucleic acid target and one or more additional oligomeric compounds targeted to a second nucleic acid target. Alternatively, compositions of the invention may contain two or more oligomeric compounds and compositions targeted to different regions, segments or sites of the same target. Two or more combined compounds may be used together or sequentially.

The formulation of therapeutic compounds and compositions of the invention and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligomeric compounds, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 μg to 100 g per kg of body weight, from 0.1 μg to 10 g per kg of body weight, from 1.0 μg to 1 g per kg of body weight, from 10.0 μg to 100 mg per kg of body weight, from 100 μg to 10 mg per kg of body weight, or from 1 mg to 5 mg per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily determine repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligomeric compound is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, from 0.1 μg to 10 g per kg of body weight, from 1 μg to 1 g per kg of body weight, from 10 μg to 100 mg per kg of body weight, from 100 μg to 10 mg per kg of body weight, or from 100 μg to 1 mg per kg of body weight, once or more daily, to once every 20 years. The effects of treatments with therapeutic compositions can be assessed following collection of tissues or fluids from a patient or subject receiving said treatments. It is known in the art that a biopsy sample can be procured from certain tissues without resulting in detrimental effects to a patient or subject. In certain embodiments, a tissue and its constituent cells comprise, but are not limited to, blood (e.g., hematopoietic cells, such as human hematopoietic progenitor cells, human hematopoietic stem cells, CD34⁺ cells CD4⁺ cells), lymphocytes and other blood lineage cells, bone marrow, breast, cervix, colon, esophagus, lymph node, muscle, peripheral blood, oral mucosa and skin. In other embodiments, a fluid and its constituent cells comprise, but are not limited to, blood, urine, semen, synovial fluid, lymphatic fluid and cerebro-spinal fluid. Tissues or fluids procured from patients can be evaluated for expression levels of a target small non-coding RNA, mRNA or protein. Additionally, the mRNA or protein expression levels of other genes known or suspected to be associated with the specific disease state, condition or phenotype can be assessed. mRNA levels can be measured or evaluated by real-time PCR, Northern blot, in situ hybridization or DNA array analysis.

Protein levels of a downstream target modulated or regulated by a small non-coding RNA can be evaluated or quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, caspase activity assays), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS). Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.

Western Blot Analysis of Protein Levels:

When small non-coding RNAs have effects on expression of downstream genes or proteins encoded by genes, it is advantageous to measure the protein levels of those gene products. To do this, western blot analysis may be employed.

Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligomeric compound treatment, washed once with PBS, suspended in Laemmli buffer (100 μl/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gradient gels (4-20%) may also be used for the separation of proteins, as is known in the art. Gels are typically run for 1.5 hours at 150 V, and transferred to a membrane, such as PVDF, for western blotting. Appropriate primary antibody directed to a target is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

Furthermore, the effects of treatment can be assessed by measuring biomarkers associated with the disease or condition in the aforementioned tissues and fluids, collected from a patient or subject receiving treatment, by routine clinical methods known in the art. These biomarkers include but are not limited to: glucose, cholesterol, lipoproteins, triglycerides, free fatty acids and other markers of glucose and lipid metabolism; liver transaminases, bilirubin, albumin, blood urea nitrogen, creatine and other markers of kidney and liver function; interleukins, tumor necrosis factors, intracellular adhesion molecules, C-reactive protein and other markers of inflammation; testosterone, estrogen and other hormones; tumor markers; vitamins, minerals and electrolytes.

In Vitro and In Vivo Assays:

Phenotypic Assays

Once modulators are designed or identified by the methods disclosed herein, the oligomeric compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive or suggestive of efficacy in the treatment, amelioration or improvement of physiologic conditions associated with a particular disease state or condition.

Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of a target in health and disease. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for a particular phenotypic assay (i.e., MCF7 cells selected for breast cancer studies; adipocytes for obesity studies) are treated with an oligomeric compound identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.

Analysis of the genotype of the cell (measurement of the expression of one or more of the genes of the cell) after treatment is also used as an indicator of the efficacy or potency of the oligomeric compound. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells.

Cell Proliferation and Survival Assays:

In some embodiments, cell proliferation and survival assays are used. Cell cycle regulation is the basis for many cancer therapeutic agents. Unregulated cell proliferation is a characteristic of cancer cells, thus most current chemotherapy agents target dividing cells, for example, by blocking the synthesis of new DNA required for cell division. However, cells in healthy tissues are often also affected by agents that modulate cell proliferation.

In some cases, a cell cycle inhibitor will cause apoptosis in cancer cells, but allow normal cells to undergo growth arrest and therefore remain unaffected (Blagosklonny, Bioessays, 1999, 21, 704-709; Chen et al., Cancer Res., 1997, 57, 2013-2019; Evan and Littlewood, Science, 1998, 281, 1317-1322; Lees and Weinberg, Proc. Natl. Acad. Sci. USA, 1999, 96, 4221-4223). An example of sensitization to anti-cancer agents is observed in cells that have reduced or absent expression of the tumor suppressor genes p53 (Bunz et al., Science, 1998, 282, 1497-1501; Bunz et al., J. Clin. Invest., 1999, 104, 263-269; Stewart et al., Cancer Res., 1999, 59, 3831-3837; Wahl et al., Nat. Med., 1996, 2, 72-79). However, cancer cells often escape apoptosis (Lowe and Lin, Carcinogenesis, 2000, 21, 485-495; Reed, Cancer J. Sci. Am., 1998, 4 Suppl 1, S8-14). Further disruption of cell cycle checkpoints in cancer cells can increase sensitivity to chemotherapy while allowing normal cells to take refuge in G1 and remain unaffected.

Cell Cycle Assay:

A cell cycle assay is employed to identify genes whose modulation affects cell cycle progression. In addition to normal cells, cells lacking functional p53 are utilized to identify genes whose modulation will sensitize p53-deficient cells to anti-cancer agents. Oligomeric compounds of the invention are tested for their effects on the cell cycle in normal human mammary epithelial cells (HMECs) as well as the breast carcinoma cell lines MCF7 and T47D. The latter two cell lines express similar genes but MCF7 cells express the tumor suppressor p53, while T47D cells are deficient in p53. A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of cell cycle progression. An oligomeric compound targeting kinesin-like 1 is known to inhibit cell cycle progression and may be used as a positive control.

Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTIN™ in OPTI-MEM™ to achieve a final concentration of 200 nM of oligomeric compound and 6 μg/mL LIPOFECTIN™. Compounds of the invention and the positive control are tested in triplicate. The negative control is tested in up to six replicate wells. Untreated control cells receive LIPOFECTIN™ only. Approximately 24, 48 or 72 hours following transfection, routine procedures are used to prepare cells for flow cytometry analysis and cells are stained with propidium iodide to generate a cell cycle profile using a flow cytometer. The cell cycle profile is analyzed with the ModFit program (Verity Software House, Inc., Topsham Me.).

Fragmentation of nuclear DNA is a hallmark of apoptosis and produces an increase in cells with a hypodiploid DNA content, which are categorized as “subG1.” An increase in cells in G1 phase is indicative of a cell cycle arrest prior to entry into S phase; an increase in cells in S phase is indicative of cell cycle arrest during DNA synthesis; and an increase in cells in the G2/M phase is indicative of cell cycle arrest just prior to or during mitosis. Cell cycle profiles of cells treated with oligomeric compounds can be normalized to those of untreated control cells, and values above or below 100% are considered to indicate an increase or decrease, respectively, in the proportion of cells in a particular phase of the cell cycle.

Oligomeric compounds that prevent cell cycle progression are candidate therapeutic agents for the treatment of hyperproliferative disorders, such as cancer or inflammation.

Caspase Assay:

Programmed cell death, or apoptosis, is an important aspect of various biological processes, including normal cell turnover, immune system development and embryonic development. Apoptosis involves the activation of caspases, a family of intracellular proteases through which a cascade of events leads to the cleavage of a select set of proteins. The caspase family can be divided into two groups: the initiator caspases, such as caspase-8 and -9, and the executioner caspases, such as caspase-3, -6 and -7, which are activated by the initiator caspases. The caspase family contains at least 14 members, with differing substrate preferences (Thomberry and Lazebnik, Science, 1998, 281, 1312-1316). A caspase assay is utilized to identify genes whose modulation causes apoptosis. The chemotherapeutic drugs taxol, cisplatin, etoposide, gemcitabine, camptothecin, aphidicolin and 5-fluorouracil all have been shown to induce apoptosis in a caspase-dependent manner.

In a further embodiment, a caspase assay is employed to identify genes or targets whose modulation affects apoptosis. In addition to normal cells, cells lacking functional p53 are utilized to identify genes or targets whose modulation will sensitize p53-deficient cells to agents that induce apoptosis. Oligomeric compounds of the invention are assayed for their affects on apoptosis in normal HMECs as well as the breast carcinoma cell lines MCF7 and T47D. HMECs and MCF7 cells express p53, whereas T47D cells do not express this tumor suppressor gene. Cells are cultured in 96-well plates with black sides and flat, transparent bottoms (Corning Incorporated, Corning, N.Y.). DMEM medium, with and without phenol red, is obtained from Invitrogen Life Technologies (Carlsbad, Calif.). MEGM medium, with and without phenol red, is obtained from Cambrex Bioscience (Walkersville, Md.). A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of caspase activity. An oligomeric compound targeted to human Jagged2 or human Notch1, both of which are known to induce caspase activity, may be used as a positive control for caspase activation.

Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTIN™ in OPTI-MEM™ to achieve a final concentration of 200 nM of oligomeric compound and 6 μg/mL LIPOFECTIN™. Compounds of the invention and the positive controls are tested in triplicate, and the negative control is tested in up to six replicate wells. Untreated control cells receive LIPOFECTIN™ only.

Caspase-3 activity is evaluated with a fluorometric HTS Caspase-3 assay (Catalog #HTS02; EMD Biosciences, San Diego, Calif.) that detects cleavage after aspartate residues in the peptide sequence DEVD. The DEVD substrate is labeled with a fluorescent molecule, which exhibits a blue to green shift in fluorescence upon cleavage by caspase-3. Active caspase-3 in the oligomeric compound-treated cells is measured by this assay according to the manufacturer's instructions. Approximately 48 hours following treatment, 50 μL of assay buffer containing 10 μM dithiothreitol is added to each well, followed by addition 20 μL of the caspase-3 fluorescent substrate conjugate. Fluorescence in wells is immediately detected (excitation/emission 400/505 nm) using a fluorescent plate reader (SpectraMAX GeminiXS, Molecular Devices, Sunnyvale, Calif.). The plate is covered and incubated at 37° C. for an additional three hours, after which the fluorescence is again measured (excitation/emission 400/505 nm). The value at time zero is subtracted from the measurement obtained at 3 hours. The measurement obtained from the untreated control cells is designated as 100% activity. Caspase-3 activity in cells treated with oligomeric compounds is normalized to that in untreated control cells. Values for caspase activity above or below 100% are considered to indicate that the compound has the ability to stimulate or inhibit caspase activity, respectively.

Oligomeric compounds that cause a significant induction in apoptosis are candidate therapeutic agents with applications in the treatment of conditions in which the induction of apoptosis is desirable, for example, in hyperproliferative disorders. Oligomeric compounds that inhibit apoptosis are candidate therapeutic agents with applications in the treatment of conditions where the reduction of apoptosis is useful, for example, in neurodegenerative disorders.

Angiogenesis Assays:

In some embodiments, angiogenesis assays are used. Angiogenesis is the growth of new blood vessels (veins and arteries) by endothelial cells. This process is important in the development of a number of human diseases, and is believed to be particularly important in regulating the growth of solid tumors. Without new vessel formation it is believed that tumors will not grow beyond a few millimeters in size. In addition to their use as anti-cancer agents, inhibitors of angiogenesis have potential for the treatment of diabetic retinopathy, cardiovascular disease, rheumatoid arthritis and psoriasis (Carmeliet and Jain, Nature, 2000, 407, 249-257; Freedman and Isner, J. Mol. Cell Cardiol, 2001, 33, 379-393; Jackson et al., Faseb J., 1997, 11, 457-465; Saaristo et al., Oncogene, 2000, 19, 6122-6129; Weber and De Bandt, Joint Bone Spine, 2000, 67, 366-383; Yoshida et al., Histol Histopathol., 1999, 14, 1287-1294).

Expression of Angiogenic Genes as a Measure of Angiogenesis:

During the process of angiogenesis, endothelial cells perform several distinct functions, including the degradation of the extracellular matrix (ECM), migration, proliferation and the formation of tube-like structures (Liekens et al., Biochem. Pharmacol, 2001, 61, 253-270). Endothelial cells must regulate the expression of many genes in order to perform the functions necessary for angiogenesis. This gene regulation has been the subject of intense scrutiny, and many genes have been identified as being important for the angiogenic phenotype. Genes highly expressed in angiogenic endothelial cells include integrin β3, endoglin/CD105, TEM5 and MMP-14/MT-MMP1.

Integrin β3 is part of a family of heterodimeric transmembrane receptors that consist of alpha and beta subunits (Brooks et al., J. Clin. Invest., 1995, 96, 1815-1822). Each subunit recognizes a unique set of ECM ligands, thereby allowing cells to transmit angiogenic signals from the extracellular matrix. Integrin β3 is prominently expressed on proliferating vascular endothelial cells, and it plays roles in allowing new blood vessels to form at tumor sites as well as allowing the epithelial cells of breast tumors to spread (Brooks et al., J. Clin. Invest., 1995, 96, 1815-1822; Drake et al., J. Cell Sci., 1995, 108 (Pt 7), 2655-2661). Blockage of integrin β3 with monoclonal antibodies or low molecular weight antagonists inhibits blood vessel formation in a variety of in-vivo models, including tumor angiogenesis and neovascularization during oxygen-induced retinopathy (Brooks et al., Science, 1994, 264, 569-571; Brooks et al., J. Clin. Invest., 1995, 96, 1815-1822; Hammes et al., Nat. Med., 1996, 2, 529-533).

Endoglin is a transforming growth factor receptor-associated protein highly expressed on endothelial cells, and present on some leukemia cells and minor subsets of bone marrow cells (Burrows et al., Clin. Cancer Res., 1995, 1, 1623-1634; Haruta and Seon, Proc. Natl. Acad. Sci. US A, 1986, 83, 7898-7902). Its expression is upregulated in endothelial cells of angiogenic tissues and is therefore used as a prognostic indicator in various tumors (Burrows et al., Clin. Cancer Res., 1995, 1, 1623-1634). Endoglin functions as an ancillary receptor influencing binding of the transforming growth factor beta (TGF-beta) family of ligands to signaling receptors, thus mediating cell survival (Massague and Chen, Genes Dev., 2000, 14, 627-644).

Tumor endothelial marker 5 (TEM5) is a putative 7-pass transmembrane protein (GPCR) (Carson-Walter et al., Cancer Res., 2001, 61, 6649-6655). The mRNA transcript, designated KIAA1531, encodes one of many tumor endothelium markers (TEMs) that display elevated expression (greater than 10-fold) during tumor angiogenesis (St Croix et al., Science, 2000, 289, 1197-1202). TEM5 is coordinately expressed with other TEMs on tumor endothelium in humans and mice.

Matrix metalloproteinase 14 (MMP-14), a membrane-type MMP covalently linked to the cell membrane, is involved in matrix detachment and migration. MMP-14 is thought to promote tumor angiogenesis; antibodies directed against the catalytic domain of MMP-14 block endothelial-cell migration, invasion and capillary tube formation in vitro (Galvez et al., J. Biol. Chem., 2001, 276, 37491-37500). MMP-14 can degrade the fibrin matrix that surrounds newly formed vessels potentially allowing the endothelial cells to invade further into the tumor tissue (Hotary et al., J. Exp. Med., 2002, 195, 295-308). MMP-14 null mice have impaired angiogenesis during development, further demonstrating the role of MMP-14 in angiogenesis (Vu and Werb, Genes Dev., 2000, 14, 2123-2133; Zhou et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 4052-4057).

In some embodiments, HUVECs are used to measure the effects of oligomeric compounds of the invention on the activity of endothelial cells stimulated with human vascular endothelial growth factor (VEGF). A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of HUVEC activity.

Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTIN™ in OPTI-MEM™ to achieve a final concentration of 75 nM of oligomeric compound and 2.25 μg/mL LIPOFECTIN™. Compounds of the invention are tested in triplicate, and the negative control is tested in up to six replicate wells. Untreated control cells receive LIPOFECTIN™ only.

Approximately twenty hours after transfection, cells are induced to express angiogenic genes with recombinant VEGF. Total RNA is harvested approximately 52 hours following transfection, and the amount of total RNA from each sample is determined using a Ribogreen Assay (Invitrogen Life Technologies, Carlsbad, Calif.). Real-time RT-PCR is performed on the total RNA using primer/probe sets for four angiogenic hallmark genes described herein: integrin β3, endoglin, TEM5 and MMP14. Expression levels for each gene are normalized to total RNA. Gene expression in cells treated with oligomeric compounds is normalized to that in untreated control cells. A value above or below 100% is considered to indicated an increase or decrease in gene expression, respectively.

Oligomeric compounds resulting in a decrease in the expression of angiogenic hallmark genes are candidate therapeutic agents for the inhibition of angiogenesis where such activity is desired, for example, in the treatment of cancer, diabetic retinopathy, cardiovascular disease, rheumatoid arthritis and psoriasis. Oligomeric compounds that increase the expression of angiogenic hallmark genes are candidate therapeutic agents with applications where the stimulation of angiogenesis is desired, for example, in wound healing.

Endothelial Tube Formation Assay as a Measure of Angiogenesis:

Angiogenesis is stimulated by numerous factors that promote interaction of endothelial cells with each other and with extracellular matrix molecules, resulting in the formation of capillary tubes. This morphogenic process is necessary for the delivery of oxygen to nearby tissues and plays an essential role in embryonic development, wound healing, and tumor growth (Carmeliet and Jain, Nature, 2000, 407, 249-257). Moreover, this process can be reproduced in a tissue culture assay that evaluated the formation of tube-like structures by endothelial cells. There are several different variations of the assay that use different matrices, such as collagen I (Kanayasu et al., Lipids, 1991, 26, 271-276), Matrigel (Yamagishi et al., J. Biol. Chem., 1997, 272, 8723-8730) and fibrin (Bach et al., Exp. Cell Res., 1998, 238, 324-334), as growth substrates for the cells. In this assay, HUVECs are plated on a matrix derived from the Engelbreth-Holm-Swarm mouse tumor, which is very similar to Matrigel (Kleinman et al., Biochemistry, 1986, 25, 312-318; Madri and Pratt, J. Histochem. Cytochem., 1986, 34, 85-91). Untreated HUVECs form tube-like structures when grown on this substrate. Loss of tube formation in vitro has been correlated with the inhibition of angiogenesis in vivo (Carmeliet and Jain, Nature, 2000, 407, 249-257; Zhang et al., Cancer Res., 2002, 62, 2034-2042), which supports the use of in vitro tube formation as an endpoint for angiogenesis.

In some embodiments, HUVECs are used to measure the effects of oligomeric compounds of the invention on endothelial tube formation activity. The tube formation assay is performed using an in vitro Angiogenesis Assay Kit (Chemicon International, Temecula, Calif.). A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of endothelial tube formation.

Oligomeric compounds are mixed with LIPOFECTIN™ in OPTI-MEM™ to achieve a final concentration of 75 nM of oligomeric compound and 2.25 μg/mL LIPOFECTIN™. Untreated control cells receive LIPOFECTIN™ only. Compounds of the invention are tested in triplicate, and the negative control is tested in up to six replicates.

Approximately fifty hours after transfection, cells are transferred to 96-well plates coated with ECMatrix™ (Chemicon International). Under these conditions, untreated HUVECs form tube-like structures. After an overnight incubation at 37° C., treated and untreated cells are inspected by light microscopy. Tube formation in cells treated with oligomeric compounds is compared to that in untreated control cells. Individual wells are assigned discrete scores from 1 to 5 depending on the extent of tube formation. A score of 1 refers to a well with no tube formation while a score of 5 is given to wells where all cells are forming an extensive tubular network.

Oligomeric compounds resulting in a decrease in tube formation are candidate therapeutic agents for the inhibition of angiogenesis where such activity is desired, for example, in the treatment of cancer, diabetic retinopathy, cardiovascular disease, rheumatoid arthritis and psoriasis. Oligomeric compounds that promote endothelial tube formation are candidate therapeutic agents with applications where the stimulation of angiogenesis is desired, for example, in wound healing.

Matrix Metalloproteinase Activity:

During angiogenesis, endothelial cells must degrade the extracellular matrix (ECM) and thus secrete matrix metalloproteinases (MMPs) in order to accomplish this degradation. MMPs are a family of zinc-dependent endopeptidases that fall into eight distinct classes: five are secreted and three are membrane-type MMPs (MT-MMPs) (Egeblad and Werb, J. Cell Science, 2002, 2, 161-174). MMPs exert their effects by cleaving a diverse group of substrates, which include not only structural components of the extracellular matrix, but also growth-factor-binding proteins, growth-factor pre-cursors, receptor tyrosine-kinases, cell-adhesion molecules and other proteinases (Xu et al., J. Cell Biol., 2002, 154, 1069-1080).

In some embodiments, oligomeric compounds of the invention are evaluated for their effects on MMP activity in the medium above cultured HUVECs. MMP activity is measured using the EnzChek Gelatinase/Collagenase Assay Kit (Molecular Probes, Eugene, Oreg.). In this assay, HUVECs are plated at approximately 4000 cells per well in 96-well plates and transfected one day later. A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of MMP activity. An oligomeric compound targeted to integrin β3 is known to inhibit MMP activity and may be used as a positive control.

Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTIN™ in OPTI-MEM™ to achieve a final concentration of 75 nM of oligomeric compound and 2.25 μg/mL LIPOFECTIN™. Compounds of the invention and the positive control are tested in triplicate, and the negative control is tested in up to six replicates. Untreated control cells receive LIPOFECTIN™ only.

Approximately 50 hours after transfection, a p-aminophenylmercuric acetate (APMA, Sigma-Aldrich, St. Louis, Mo.) solution is added to each well of a Corning-Costar 96-well clear bottom plate (VWR International, Brisbane, Calif.). The APMA solution is used to promote cleavage of inactive MMP precursor proteins. Medium above the HUVECs is then transferred to the wells in the 96-well plate. After approximately 30 minutes, the quenched, fluorogenic MMP cleavage substrate is added, and baseline fluorescence is read immediately at 485 nm excitation/530 nm emission. Following an overnight incubation at 37° C. in the dark, plates are read again to determine the amount of fluorescence, which corresponds to MMP activity. Total protein from HUVEC lysates is used to normalize the readings, and MMP activity from cells treated with oligomeric compounds is normalized to that of untreated control cells. MMP activities above or below 100% are considered to indicate a stimulation or inhibition, respectively, of MMP activity.

Oligomeric compounds resulting in a decrease in MMP activity are candidate therapeutic agents for the inhibition of angiogenesis where such activity is desired, for example, in the treatment of cancer, diabetic retinopathy, cardiovascular disease, rheumatoid arthritis and psoriasis. Oligomeric compounds that increase the expression of angiogenic hallmark genes are candidate therapeutic agents with applications in conditions requiring angiogenesis, for example, in wound healing.

Adipocyte Assays:

In some embodiments, adipocytes assays are used. Insulin is an essential signaling molecule throughout the body, but its major target organs are the liver, skeletal muscle and adipose tissue. Insulin is the primary modulator of glucose homeostasis and helps maintain a balance of peripheral glucose utilization and hepatic glucose production. The reduced ability of normal circulating concentrations of insulin to maintain glucose homeostasis manifests in insulin resistance which is often associated with diabetes, central obesity, hypertension, polycystic ovarian syndrome, dyslipidemia and atherosclerosis (Saltiel, Cell, 2001, 104, 517-529; Saltiel and Kahn, Nature, 2001, 414, 799-806).

Response of Undifferentiated Adipocytes to Insulin:

Insulin promotes the differentiation of preadipocytes into adipocytes. The condition of obesity, which results in increases in fat cell number, occurs even in insulin-resistant states in which glucose transport is impaired due to the antilipolytic effect of insulin. Inhibition of triglyceride breakdown requires much lower insulin concentrations than stimulation of glucose transport, resulting in maintenance or expansion of adipose stores (Kitamura et al., Mol Cell Biol., 1999, 19, 6286-6296; Kitamura et al., Mol Cell Biol., 1998, 18, 3708-3717).

One of the hallmarks of cellular differentiation is the upregulation of gene expression. During adipocyte differentiation, the gene expression patterns in adipocytes change considerably. Some genes known to be upregulated during adipocyte differentiation include hormone-sensitive lipase (HSL), adipocyte lipid binding protein (aP2), glucose transporter 4 (Glut4), and peroxisome proliferator-activated receptor gamma (PPAR-γ). Insulin signaling is improved by compounds that bind and inactivate PPAR-Y, a key regulator of adipocyte differentiation (Olefsky, J. Clin. Invest., 2000, 106, 467-472). Insulin induces the translocation of GLUT4 to the adipocyte cell surface, where it transports glucose into the cell, an activity necessary for triglyceride synthesis. In all forms of obesity and diabetes, a major factor contributing to the impaired insulin-stimulated glucose transport in adipocytes is the downregulation of GLUT4. Insulin also induces hormone sensitive lipase (HSL), which is the predominant lipase in adipocytes that functions to promote fatty acid synthesis and lipogenesis (Fredrikson et al., J. Biol. Chem., 1981, 256, 6311-6320). Adipocyte fatty acid binding protein (aP2) belongs to a multi-gene family of fatty acid and retinoid transport proteins. aP2 is postulated to serve as a lipid shuttle, solubilizing hydrophobic fatty acids and delivering them to the appropriate metabolic system for utilization (Fu et al., J. Lipid Res., 2000, 41, 2017-2023; Pelton et al., Biochem. Biophys. Res. Commun., 1999, 261, 456-458). Together, these genes play important roles in the uptake of glucose and the metabolism and utilization of fats.

Leptin secretion and an increase in triglyceride content are also well-established markers of adipocyte differentiation. In addition to its role in adipocytes differentiation, leptin also regulates glucose homeostasis through mechanisms (autocrine, paracrine, endocrine and neural) independent of the adipocyte's role in energy storage and release. As adipocytes differentiate, insulin increases triglyceride accumulation by both promoting triglyceride synthesis and inhibiting triglyceride breakdown (Spiegelman and Flier, Cell, 2001, 104, 531-543). As triglyceride accumulation correlates tightly with cell size and cell number, it is an excellent indicator of differentiated adipocytes.

Oligomeric compounds of the invention are tested for their effects on preadipocyte differentiation. A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of adipocyte differentiation. Tumor necrosis factor alpha (TNF-α) is known to inhibit adipocyte differentiation and may be used as a positive control for the inhibition of adipocyte differentiation as evaluated by leptin secretion. For the other adipocyte differentiation markers assayed, an oligomeric compound targeted to PPAR-γ, also known to inhibit adipocyte differentiation, may be used as a positive control.

Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTIN™ in OPTI-MEM™ to achieve a final concentration of 250 nM of oligomeric compound and 7.5 μg/mL LIPOFECTIN™. Untreated control cells receive LIPOFECTIN™ only. Compounds of the invention and the positive control are tested in triplicate, and the negative control is tested in up to six replicate wells.

After the cells have reach confluence (approximately three days), they are exposed for an additional three days to differentiation medium (Zen-Bio, Inc., Research Triangle Park, N.C.) containing a PPAR-γ agonist, IBMX, dexamethasone, and insulin. Cells are then fed adipocyte medium (Zen-Bio, Inc.), which is replaced at 2 or 3 day intervals.

Leptin secretion into the medium in which adipocytes are cultured is measured by protein ELISA. On day nine post-transfection, 96-well plates are coated with a monoclonal antibody to human leptin (R&D Systems, Minneapolis, Minn.) and left at 4° C. overnight. The plates are blocked with bovine serum albumin (BSA), and a dilution of the treated adipoctye medium is incubated in the plate at room temperature for approximately 2 hours. After washing to remove unbound components, a second monoclonal antibody to human leptin (conjugated with biotin) is added. The plate is then incubated with strepavidin-conjugated horse radish peroxidase (HRP) and enzyme levels are determined by incubation with 3,3′,5,5′-tetramethlybenzidine, which turns blue when cleaved by HRP. The OD₄₅₀ is read for each well, where the dye absorbance is proportional to the leptin concentration in the cell lysate. Leptin secretion from cells treated with oligomeric compounds is normalized to that from untreated control cells. With respect to leptin secretion, values above or below 100% are considered to indicate that the compound has the ability to stimulate or inhibit leptin secretion, respectively.

The triglyceride accumulation assay measures the synthesis of triglyceride by adipocytes. Triglyceride accumulation is measured using the Infinity™ Triglyceride reagent kit (Sigma-Aldrich, St. Louis, Mo.). On day nine post-transfection, cells are washed and lysed at room temperature, and the triglyceride assay reagent is added. Triglyceride accumulation is measured based on the amount of glycerol liberated from triglycerides by the enzyme lipoprotein lipase. Liberated glycerol is phosphorylated by glycerol kinase, and hydrogen peroxide is generated during the oxidation of glycerol-1-phosphate to dihydroxyacetone phosphate by glycerol phosphate oxidase. Horseradish peroxidase (HRP) uses H₂O₂ to oxidize 4-aminoantipyrine and 3,5 dichloro-2-hydroxybenzene sulfonate to produce a red-colored dye. Dye absorbance, which is proportional to the concentration of glycerol, is measured at 515 nm using an UV spectrophotometer. Glycerol concentration is calculated from a standard curve for each assay, and data are normalized to total cellular protein as determined by a Bradford assay (Bio-Rad Laboratories, Hercules, Calif.). Triglyceride accumulation in cells treated with oligomeric compounds is normalized to that in untreated control cells. Values for triglyceride accumulation above or below 100% are considered to indicate that the compound has the ability to stimulate or inhibit triglyceride accumulation, respectively.

Expression of the four hallmark genes, HSL, aP2, Glut4, and PPARγ, is also measured in adipocytes transfected with oligomeric compounds of the invention. Cells are lysed on day nine post-transfection and total RNA is harvested. The amount of total RNA in each sample is determined using a Ribogreen Assay (Invitrogen Life Technologies, Carlsbad, Calif.). Real-time PCR is performed on the total RNA using primer/probe sets for the adipocyte differentiation hallmark genes Glut4, HSL, aP2, and PPAR-Y. Gene expression in cells treated with oligomeric compounds is normalized to that in untreated control cells. With respect to the four adipocyte differentiation hallmark genes, values above or below 100% are considered to indicate that the compound has the ability to stimulate or inhibit adipocyte differentiation, respectively.

Oligomeric compounds that reduce the expression levels of markers of adipocyte differentiation are candidate therapeutic agents with applications in the treatment, attenuation or prevention of obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases as well as having potential applications in the maintenance of the pluripotent phenotype of stem or precursor cells. Oligomeric compounds of the invention resulting in a significant increase in leptin secretion are potentially useful for the treatment of obesity.

Response of Liver-Derived Cells to Insulin:

Insulin mediates its effects by suppressing the RNA expression levels of enzymes important for gluconeogenesis and glycogenolysis, and also by controlling the activities of some metabolic enzymes through post-translational mechanisms (Hall and Granner, J. Basic Clin. Physiol Pharmacol, 1999, 10, 119-133; Moller, Nature, 2001, 414, 821-827; Saltiel and Kahn, Nature, 2001, 414, 799-806). In liver cells, genes involved in regulating glucose metabolism can be identified by monitoring changes in the expression of selective insulin-responsive genes in a cell culture model. However, primary human hepatocytes are difficult to obtain and work with in culture. Therefore, the insulin signaling assay described herein is performed in the hepatocellular carcinoma cell line HepG2, the most widely used cell culture model for hepatocytes. The insulin responsive genes evaluate in this assay are phosphoenolpyruvate carboxykinase (PEPCK), insulin-like growth factor binding protein 1 (IGFBP-1) and follistatin.

IGFBP-1 is one of a family of six secreted proteins that bind insulin-like growth factor (IGF) with high affinity and thereby modulate IGFs action in vivo (Baxter, Am. J. Physiol Endocrinol Metab., 2000, 278, E967-976; Lee et al., Proc. Soc. Exp. Biol. Med., 1997, 216, 319-357). IGFBP-1 is characterized by dynamic variability of levels in circulation due to the regulation of its hepatic secretion (Lee et al., Proc. Soc. Exp. Biol. Med., 1997, 216, 319-357). The multi-hormonal regulation of PEPCK and IGFBP-1 are similar. Glucocorticoids and cyclic AMP (cAMP) stimulate transcription of the IGFBP-1 gene expression whereas insulin acts in a dominant manner to suppress both basal and cAMP or glucocorticoid-stimulated IGFBP-1 gene transcription (O'Brien and Granner, Physiol. Rev., 1996, 76, 1109-1161). PEPCK catalyzes the rate-limiting step in gluconeogenesis, and thereby contributes to hepatic glucose output (Hall and Granner, J. Basic Clin. Physiol Pharmacol, 1999, 10, 119-133; Moller, Nature, 2001, 414, 821-827; Saltiel and Kahn, Nature, 2001, 414, 799-806). In hepatoma cells, studies have shown that the expression of PEPCK is stimulated by glucocorticoids, glucagon (via cAMP), and retinoic acid. Insulin acts in a dominant manner to suppress these stimulations as well as basal transcription (O'Brien and Granner, Physiol Rev., 1996, 76, 1109-1161). In HepG2 cells, prolonged serum starvation induces the expression of PEPCK and subsequent insulin stimulation significantly reduces the PEPCK mRNA level.

Follistatin is significantly stimulated by insulin in HepG2 cells. Interestingly, follistatin levels have been shown to be higher in women with polycystic ovary syndrome (PCOS) (Norman et al., Hum. Reprod., 2001, 16, 668-672). PCOS is a metabolic as well as a reproductive disorder, and an important cause of type 2 diabetes mellitus in women. It is often associated with profound insulin resistance and hyperinsulinemia as well as with a defect in insulin secretion (Dunaif, Endocr. Rev., 1997, 18, 774-800; Nestler et al., Fertil Steril, 2002, 77, 209-215). PCOS is the most common cause of female infertility in the U.S. and affects 5%-10% of women of child-bearing age (Dunaif, Endocr. Rev., 1997, 18, 774-800; Nestler et al., Fertil Steril, 2002, 77, 209-215).

In some embodiments, HepG2 cells are used to measure the effects of compounds of the invention on hepatic gene expression following insulin stimulation. A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of hepatic gene expression. Insulin at a concentration of 100 nM may be used as a positive control for the stimulation of hepatic gene expression. An oligomeric compound targeted to human forkhead is known to inhibit hepatic gene expression and may be used as a positive control for the inhibition of gene expression in the presence of insulin.

Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTIN™ in OPTI-MEM™ to achieve a final concentration of 100 nM of oligomeric compound and 3 μg/mL LIPOFECTIN™. Untreated control cells receive LIPOFECTIN™ only. Compounds of the invention and the positive controls are tested in triplicate, and the negative control is tested in up to six replicate wells.

Approximately 28 hours after transfection, the cells are subjected to serum starvation for a period of 12 to 16 hours, using serum-free growth medium. Following serum starvation, cells are treated with 1 nM insulin (insulin-treated) or are left untreated (basal conditions) for approximately four hours. At the same time, untreated control cells in both plates are treated with 100 nM insulin to determine the maximal insulin response. Following insulin treatment (forty-eight hours after transfection), total RNA is harvested from all samples, and the amount of total RNA from each sample is determined using a Ribogreen assay (Invitrogen Corporation, Carlsbad, Calif.). Real-time PCR is performed on the total RNA samples using primer/probe sets for three insulin responsive genes: insulin-like growth factor binding protein-1 (IGFBP-1), cytosolic PEPCK (PEPCK-C), and follistatin. Gene expression levels obtained by real-time PCR are normalized for total RNA content in the samples. Gene expression in cells treated with oligomeric compounds is normalized to that from untreated control cells. Values above or below 100% are considered to indicate an increase or decrease in gene expression, respectively.

Oligomeric compounds that interfere with the expression of genes involved in glucose metabolism are candidate therapeutic agents for the treatment of conditions associated with abnormal glucose metabolism, for example, obesity and diabetes.

Inflammation Assays:

In some embodiments, inflammation assays are used. Inflammation assays are designed to identify genes that regulate the activation and effector phases of the adaptive immune response. During the activation phase, T lymphocytes (also known as T-cells) receiving signals from the appropriate antigens undergo clonal expansion, secrete cytokines, and up-regulate their receptors for soluble growth factors, cytokines and co-stimulatory molecules (Cantrell, Annu. Rev. Immunol, 1996, 14, 259-274). These changes drive T-cell differentiation and effector function. Response to cytokines by non-immune effector cells controls the production of inflammatory mediators that can do extensive damage to host tissues. The cells of the adaptive immune systems, their products, as well as their interactions with various enzyme cascades involved in inflammation (e.g., the complement, clotting, fibrinolytic and kinin cascades) all represent potential points for intervention in inflammatory disease.

Dendritic cells treated with oligomeric compounds targeting different genes are used to identify regulators of dendritic cell-mediated T-cell co-stimulation. The level of interleukin-2 (IL-2) production by T-cells, a critical consequence of T-cell activation (DeSilva et al., J. Immunol., 1991, 147, 3261-3267; Salomon and Bluestone, Annu. Rev. Immunol., 2001, 19, 225-252), is used as an endpoint for T-cell activation. T lymphocytes are important immunoregulatory cells that mediate pathological inflammatory responses. Optimal activation of T lymphocytes requires both primary antigen recognition events as well as secondary or co-stimulatory signals from antigen presenting cells (APC). Dendritic cells are the most efficient APCs known and are principally responsible for antigen presentation to T-cells, expression of high levels of co-stimulatory molecules during infection and disease, and the induction and maintenance of immunological memory (Banchereau and Steinman, Nature, 1998, 392, 245-252). While a number of co-stimulatory ligand-receptor pairs have been shown to influence T-cell activation, a principal signal is delivered by engagement of CD28 on T-cells by CD80 (B7-1) and CD86 (B7-2) on APCs (Boussiotis et al., Curr. Opin. Immunol, 1994, 6, 797-807; Lenschow et al., Annu. Rev. Immunol, 1996, 14, 233-258). In contrast, a B7 counter-receptor, CTLA-4, has been shown to negatively regulate T-cell activation, maintain immunological homeostasis and promote immune tolerance (Walunas and Bluestone, J. Immunol., 1998, 160, 3855-3860). Inhibition of T-cell co-stimulation by APCs holds promise for novel and more specific strategies of immune suppression. In addition, blocking co-stimulatory signals may lead to the development of long-term immunological anergy (unresponsiveness or tolerance) that would offer utility for promoting transplantation or dampening autoimmunity. T-cell anergy is the direct consequence of failure of T-cells to produce the growth factor interleukin-2 (DeSilva et al., J. Immunol., 1991, 147, 3261-3267; Salomon and Bluestone, Annu. Rev. Immunol., 2001, 19, 225-252). Dendritic cell cytokine production as a measure of the activation phase of the immune response:

In some embodiments, the effects of the oligomeric compounds of the invention are examined on the dendritic cell-mediated costimulation of T-cells. A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of dendritic cell-mediated T-cell costimulation. An oligomeric compound targeted to human CD86 is known to inhibit dendritic cell-mediated T-cell stimulation and may be used as a positive control.

Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTIN™ in OPTI-MEM™ to achieve a final concentration of 200 nM of oligomeric compound and 6 μg/mL LIPOFECTIN™. Untreated control cells receive LIPOFECTIN™ only. Compounds of the invention and the positive control are tested in triplicate, and the negative control is tested in up to six replicates. Following incubation with the oligomeric compounds and LIPOFECTIN™, fresh growth medium with cytokines is added and DC culture is continued for an additional 48 hours. DCs are then co-cultured with Jurkat T-cells in RPMI medium (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% heat-inactivated fetal bovine serum (Sigma Chemical Company, St. Louis, Mo.). Culture supernatants are collected 24 hours later and assayed for IL-2 levels (IL-2 DuoSet, R&D Systems, Minneapolis, Minn.). IL-2 levels in cells treated with oligomeric compounds are normalized to those from untreated control cells. A value greater than 100% indicates an induction of the inflammatory response, whereas a value less than 100% demonstrates a reduction in the inflammatory response.

Oligomeric compounds that inhibit T-cell co-stimulation are candidate therapeutic compounds with applications in the prevention, treatment or attenuation of conditions associated with hyperstimulation of the immune system, including rheumatoid arthritis, irritable bowel disease, asthma, lupus and multiple sclerosis. Oligomeric compounds that induce T-cell co-stimulation are candidate therapeutic agents for the treatment of immunodeficient conditions.

Cytokine Signaling as a Measure of the Effector Phase of the Inflammatory Response:

The cytokine signaling assay further identifies genes that regulate inflammatory responses of non-immune effector cells (initially endothelial cells) to stimulation with cytokines such as interferon-gamma (IFN-γ). Response to IFN-γ is assessed by measuring the expression levels of three genes: intercellular adhesion molecule-1 (ICAM-1), interferon regulatory factor 1 (IRF1) and small inducible cytokine subfamily B (Cys-X-Cys), member 11 (SCYB11). The cytokine signaling assay further identifies genes that regulate inflammatory responses of non-immune effector cells (initially endothelial cells) to stimulation with IL-1β or TNF-α (Heyninck et al., J Cell Biol, 1999, 145, 1471-1482; Zetoune et al., Cytokine, 2001, 15, 282-298). Response to IL-1β or TNF-α stimulation is monitored by measuring the expression levels of four genes: A20, intracellular adhesion molecule 1 (ICAM-1), interleukin-9 (IL-8) and macrophage-inflammatory protein 2 (MIP2α). As described below, all of these genes regulate numerous parameters of the inflammatory response.

ICAM-1 is an adhesion molecule expressed at low levels on resting endothelial cells that is markedly up-regulated in response to inflammatory mediators like tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interferon-γ (IFN-γ) (Springer, Nature, 1990, 346, 425-434). ICAM-1 expression serves to attract circulating leukocytes into the inflammatory site.

IRF-1 binds to upstream cis-regulatory elements of interferon-inducible genes and functions as a transcriptional activator. IRF-1 directly binds to a functional IFN-γ-stimulated response element in the cathepsin S promoter and mediates IFN-γ dependent transcriptional activation (Storm van's Gravesande et al., J Immunol., 2002, 168, 4488-4494).

SCYB11 is essential for mediating normal leukocyte recruitment and trafficking during inflammation. SCYB11 induces a chemotactic response in IL-2 activated T-cells, monocytes and granulocytes (Mohan et al., J Immunol., 2002, 168, 6420-6428).

A20 is a zinc-finger protein that limits the transcription of pro-inflammatory genes by blocking TRAF2-stimulated NK-κB signaling. Studies in mice show that TNF-α dramatically increases A20 expression in mice, and that A20 expression is crucial for their survival (Lee et al., Science, 2000, 289, 2350-2354).

IL-8 is a member of the chemokine gene superfamily, members of which promote the pro-inflammatory phenotype of macrophages, vascular smooth muscle cells and endothelial cells (Koch et al., Science, 1992, 258, 1798-1801). IL-8 has been known as one of the major inducible chemokines with the ability to attract neutrophils to the site of inflammation. More recently, IL-8 has been implicated as a major mediator of acute neutrophil-mediated inflammation, and is therefore a potential anti-inflammatory target (Mukaida et al., Cytokine Growth Factor Rev, 1998, 9, 9-23).

MIP2α, another chemokine known to play a central role in leukocyte extravasation, has more recently been shown to be involved in acute inflammation (Lukacs et al., Chem Immunol., 1999, 72, 102-120). MIP2α is expressed in response to microbial infection, to injection of lipopolysaccharides (LPS), and to stimulation of cells with pro-inflammatory mediators such as IL-1β and TNF-α (Kopydlowski et al., J Immunol., 1999, 163, 1537-1544). Endothelial cells are one of several cell types that are sources of MIP2α (Rudner et al., J Immunol., 2000, 164, 6576-6582).

In some embodiments, the effects of the oligomeric compounds of the invention on the cellular response to cytokines may be examined in HUVECs. A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of cytokine signaling.

Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTIN™ in OPTI-MEM™ to achieve a final concentration of 75 nM of oligomeric compound and 2.25 μg/mL LIPOFECTIN™. Untreated control cells receive LIPOFECTIN™ only. Compounds of the invention are tested in triplicate, and the negative control is tested in up to six replicate wells.

For IFN-γ stimulation, following transfection, fresh growth medium is added and DC culture is continued for an additional 44 hours, after which HUVECS are stimulated with 10 ng/ml of IFN-γ for a period of 4 hours. For stimulation with IL-1β or TNF-α, fresh growth medium is added and DC culture is continued for an additional 46 hours, after which HUVECs are stimulated with 0.1 ng/mL of IL-1β or 1 ng/mL of TNF-α for a period of 2 hours. Total RNA is harvested 48 hours following transfection, and real time PCR is performed using primer/probe sets to detect ICAM-1, IRF-1 and SCYB11 in IFN-γ-stimulated cells, or ICAM-1, A20, IL-8 and MIP2α in IL-1β-stimulated and TNF-α-stimulated cells. Expression levels of each gene are normalized to total RNA. Gene expression levels from cells treated with oligomeric compounds are normalized to those from untreated control cells. A value greater than 100% indicates an induction of the inflammatory response, whereas a value less than 100% demonstrates a reduction in the inflammatory response.

Oligomeric compounds that inhibit the inflammatory response are candidate therapeutic compounds with applications in the prevention, treatment or attenuation of conditions associated with hyperstimulation of the immune system, including rheumatoid arthritis, irritable bowel disease, asthma, lupus and multiple sclerosis.

In Vivo Studies

The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.

Mouse Model of Tumorigenesis:

Animal models of tumorigenesis are used in some embodiments of the present invention. In this model, tumorigenic cells are injected into immunocompromised mice (i.e. nude mice), and subsequent growth of a tumor is measured.

Serially transplanted MDA-MB-231 (a human breast carcinoma cell line, American Type Culture Collection, Manassus, Va.) tumors are established subcutaneously in nude mice. Beginning two weeks later, one or more of the oligomeric compounds of the invention are administered intravenously daily for 14 days at dosages of 15 mg/kg or 30 mg/kg. Control compounds are also administered at these doses, and a saline control is also given. Tumor growth rates are monitored for the two-week period of oligonucleotide administration. Activity of the oligomeric compounds of the invention is measured by a reduction in tumor growth. Activity is measured by reduced tumor volume compared to saline or control compound. Following death or sacrifice of mice, tumor tissue is fixed in 4% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin. Tumor tissue sections are evaluated for tumor morphology and size.

Human A549 lung tumor cells are also injected into nude mouse to produce tumors. 200 μl of A549 cells (5×10⁶ cells) are implanted subcutaneously in the inner thigh of nude mice. Oligomeric compounds of the invention are administered twice weekly for four weeks, beginning one week following tumor cell inoculation. Oligomeric compounds are formulated with cationic lipids (LIPOFECTIN™, Invitrogen Corporation, Carlsbad, Calif.) and given subcutaneously in the vicinity of the tumor. Oligomeric compound dosage is 5 mg/kg with 60 mg/kg cationic lipid. Tumor size is recorded weekly. Activity of the oligomeric compounds of the invention is measured by reduction in tumor size compared to controls.

Xenograft studies are also performed using the U-87 human glioblastoma cell line (American Type Culture Collection, Manassus, Va.). Nude mice are injected subcutaneously with 2×10⁷ U-87 cells. Mice are injected intraperitoneally with one or more of the oligomeric compounds of the invention or a control compound at dosages of either 15 mg/kg or 30 mg/kg for 21 consecutive days beginning 7 days after xenografts are implanted. Saline-injected animals serve as a control. Tumor volumes are measured on days 14, 21, 24, 31 and 35. Activity is measured by reduced tumor volume compared to saline or control compound. Following death or sacrifice of mice, tumor tissue is fixed in 4% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin. Tumor tissue sections are evaluated for tumor morphology and size.

Alternatively, intracerebral U-87 xenografts are generated by implanting U-87 glioblastoma cells into the brains of nude mice. Mice are treated via continuous intraperitoneal administration with one or more of the oligomeric compounds of the invention at 20 mg/kg, control compound at 20 mg/kg or saline beginning on day 7 after xenograft implantation. Activity of the oligomeric compounds of the invention is measured by an increase in survival time compared to controls. Following death or sacrifice, brain tissue is fixed in 4% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin. Brain tissue sections are evaluated for tumor growth.

Leptin-Deficient Mice (a Model of Obesity and Diabetes (ob/ob Mice)):

Leptin is a hormone produced by fat cells that regulates appetite. Deficiencies in this hormone in both humans and non-human animals leads to obesity. ob/ob mice have a mutation in the leptin gene which results in obesity and hyperglycemia. As such, these mice are a useful model for the investigation of obesity and diabetes and treatments designed to treat these conditions. ob/ob mice have higher circulating levels of insulin and are less hyperglycemic than db/db mice, which harbor a mutation in the leptin receptor. In accordance with the present invention, the oligomeric compounds of the invention are tested in the ob/ob model of obesity and diabetes.

Seven-week old male C57B1/6J-Lep ob/ob mice (Jackson Laboratory, Bar Harbor, Me.) are fed a diet with a fat content of 10-15% and are subcutaneously injected with the oligomeric compounds of the invention or a control compound at a dose of 25 mg/kg two times per week for 4 weeks. Saline-injected animals, leptin wildtype littermates (i.e. lean littermates) and ob/ob mice fed a standard rodent diet serve as controls. After the treatment period, mice are sacrificed and target levels are evaluated in liver, brown adipose tissue (BAT) and white adipose tissue (WAT). RNA isolation and target RNA expression level quantitation are performed as described by other examples herein.

To assess the physiological effects resulting from modulation of target, the ob/ob mice are evaluated at the end of the treatment period for serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis, or clearing of lipids from the liver, is assessed by measuring the liver triglyceride content. Hepatic steatosis is assessed by routine histological analysis of frozen liver tissue sections stained with oil red O stain, which is commonly used to visualize lipid deposits, and counterstained with hematoxylin and eosin, to visualize nuclei and cytoplasm, respectively.

The effects of target modulation on glucose and insulin metabolism are evaluated in the ob/ob mice treated with the oligomeric compounds of the invention. Plasma glucose is measured at the start of the treatment and after 2 weeks and 4 weeks of treatment. Plasma insulin is similarly measured at the beginning of the treatment, and following at 2 weeks and at 4 weeks of treatment. Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose and insulin levels are measured before the insulin or glucose challenge and at 15, 20 or 30 minute intervals for up to 3 hours.

To assess the metabolic rate of ob/ob mice treated with the oligomeric compounds of the invention, the respiratory quotient and oxygen consumption of the mice are also measured.

The ob/ob mice that receive treatment are evaluated at the end of the treatment period for the effects of target modulation on the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism. These genes include, but are not limited to, HMG-CoA reductase, acetyl-CoA carboxylase 1 and acetyl-CoA carboxylase 2, carnitine palmitoyltransferase I and glycogen phosphorylase, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase 1, lipoprotein lipase and hormone sensitive lipase. mRNA levels in liver and white and brown adipose tissue are quantitated by real-time PCR as described in other examples herein, employing primer/probe sets that are generated using published sequences of each gene of interest.

Leptin Receptor-Deficient Mice (a Model of Obesity and Diabetes (db/db Mice)):

db/db mice have a mutation in the leptin receptor gene which results in obesity and hyperglycemia. As such, these mice are a useful model for the investigation of obesity and diabetes and treatments designed to treat these conditions. db/db mice, which have lower circulating levels of insulin and are more hyperglycemic than ob/ob mice which harbor a mutation in the leptin gene, are often used as a rodent model of type 2 diabetes. In accordance with the present invention, oligomeric compounds of the present invention are tested in the db/db model of obesity and diabetes.

Seven-week old male C57B1/6J-Lepr db/db mice (Jackson Laboratory, Bar Harbor, Me.) are fed a diet with a fat content of 15-20% and are subcutaneously injected with one or more of the oligomeric compounds of the invention or a control compound at a dose of 25 mg/kg two times per week for 4 weeks. Saline-injected animals, leptin receptor wildtype littermates (i.e. lean littermates) and db/db mice fed a standard rodent diet serve as controls. After the treatment period, mice are sacrificed and target levels are evaluated in liver, BAT and WAT as described supra.

To assess the physiological effects resulting from modulation of target, the db/db mice that receive treatment are evaluated at the end of the treatment period for serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis is assessed by measuring the liver triglyceride content and oil red O staining, as described supra.

The effects of target modulation on glucose and insulin metabolism are also evaluated in the db/db mice treated with the oligomeric compounds of the invention. Plasma glucose is measured at the start of the treatment and after 2 weeks and 4 weeks of treatment. Plasma insulin is similarly measured at the beginning of the treatment, and following 2 weeks and 4 weeks of treatment. Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose levels are measured before the insulin or glucose challenge and 15, 30, 60, 90 and 120 minutes following the injection.

To assess the metabolic rate of db/db mice treated with the oligomeric compounds of the invention, the respiratory quotient and oxygen consumption of the mice is also measured.

The db/db mice that receive treatment are evaluated at the end of the treatment period for the effects of target modulation on the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism, as described supra.

Lean Mice on a Standard Rodent Diet:

C57B1/6 mice are maintained on a standard rodent diet and are used as control (lean) animals. In one embodiment of the present invention, the oligomeric compounds of the invention are tested in normal, lean animals.

Seven-week old male C57B1/6 mice are fed a diet with a fat content of 4% and are subcutaneously injected with one or more of the oligomeric compounds of the invention or control compounds at a dose of 25 mg/kg two times per week for 4 weeks. Saline-injected animals serve as a control. After the treatment period, mice are sacrificed and target levels are evaluated in liver, BAT and WAT as described supra.

To assess the physiological effects resulting from modulation of the target, the lean mice that receive treatment are evaluated at the end of the treatment period for serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis is assessed by measuring the liver triglyceride content and oil red 0 staining, as described supra.

The effects of target modulation on glucose and insulin metabolism are also evaluated in the lean mice treated with the oligomeric compounds of the invention. Plasma glucose is measured at the start of the treatment and after 2 weeks and 4 weeks of treatment. Plasma insulin is similarly measured at the beginning of the treatment, and following 2 weeks and 4 weeks of treatment. Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose levels are measured before the insulin or glucose challenge and 15, 30, 60, 90 and 120 minutes following the injection.

To assess the metabolic rate of lean mice treated with the oligomeric compounds of the invention, the respiratory quotient and oxygen consumption of the mice is also measured.

The lean mice that received treatment are evaluated at the end of the treatment period for the effects of target modulation on the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism, as described supra.

Levin Model of Diet-Induced Obesity in Rats:

The Levin Model is a polygenic model of rats selectively bred to develop diet-induced obesity (DIO) associated with impaired glucose tolerance, dyslipidemia and insulin resistance when fed a high-fat diet (Levin, et al., Am. J. Physiol, 1997, 273, R725-30). The advantage of this model is that it displays traits more similar to human obesity and glucose intolerance than in animals that are obese/hyperinsulinemic due to genetic defects e.g. defects in leptin signaling. This model is useful in investigating the oligomeric compounds of the present invention for their ability to affect obesity and related complications, such as impaired glucose tolerance, dyslipidemia and insulin resistance. In accordance with the present invention, the oligomeric compounds of the invention are tested in the Levin model of diet-induced obesity.

Eight-week old male Levin rats (Charles River Laboratories, Wilmington, Mass.), weighing ˜500 g, are fed a diet with a fat content of 60% for eight weeks, after which they are subcutaneously injected with one or more of the oligomeric compounds of the invention at a dose of 25 mg/kg×2 per week for 8 weeks. Control groups consist of animals injected with saline or a control compound and lean littermates fed a standard rodent diet. The control compound is injected at the same dose as the target-specific compound.

Throughout the treatment period, the rats are evaluated for food consumption, weight gain, as well as serum levels of glucose, insulin, cholesterol, free fatty acids, triglycerides and liver enzymes.

The effects of target modulation on glucose and insulin metabolism are also evaluated in the Levin rats treated with the oligomeric compounds of the invention. Plasma glucose and insulin are monitored throughout the treatment by analyzing blood samples. Glucose and tolerance are assessed in fed or fasted rats. After blood is collected for baseline glucose and insulin levels, a glucose challenge is administered, after which blood glucose and insulin levels are measured at 15, 20 or 30 minute intervals for up to 3 hours. Insulin tolerance is similarly analyzed, beginning with blood collection for baseline glucose and insulin levels, followed by an insulin challenge, after which blood glucose levels are measured at 15, 20 or 30 minute intervals for up to 3 hours. Plasma insulin and glucose are also measured at study termination.

At the end of the treatment period, the rats are sacrificed. Organs are removed and weighed, including liver, white adipose tissue, brown adipose tissue and spleen. Target RNA expression levels are measured in all tissues that are isolated, using quantitative real-time PCR. Target protein levels are also evaluated by immunoblot analysis using antibodies that specifically recognize the target protein.

Also evaluated at the end of the treatment period are serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis is assessed by measuring the liver triglyceride content and oil red 0 staining, as described supra.

The Levin rats that receive treatment are evaluated at the end of the treatment period for the effects of target modulation on the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism, as described supra.

C57BL/6 on a High-Fat Diet (A Model of Diet-Induced Obesity (DIO)):

The C57BL/6 mouse strain is reported to be susceptible to hyperlipidemia-induced atherosclerotic plaque formation. Consequently, when these mice are fed a high-fat diet, they develop diet-induced obesity. Accordingly these mice are a useful model for the investigation of obesity and treatments designed to treat these conditions. In one embodiment of the present invention, the oligomeric compounds of the invention are tested in a model of diet-induced obesity.

Male C57BL/6 mice (7-weeks old) receive a 60% fat diet for 8 weeks, after which mice are subcutaneously injected with one or more of the oligomeric compounds of the invention at a dose of 25 mg/kg two times per week for 4 weeks. Saline-injected or control compound-injected animals serve as a control. After the treatment period, mice are sacrificed and target levels are evaluated in liver, BAT and WAT as described supra.

To assess the physiological effects resulting from modulation of target, the diet-induced obese mice that receive treatment are evaluated at the end of the treatment period for serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis is assessed by measuring the liver triglyceride content and oil red O staining, as described supra.

The effects of target modulation on glucose and insulin metabolism are also evaluated in the diet-induced obese mice treated with the oligomeric compounds of the invention. Plasma glucose is measured at the start of treatment and after 2 weeks and 4 weeks of treatment. Plasma insulin is similarly measured at the beginning of the treatment, and following 2 weeks and 4 weeks of treatment. Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose and insulin levels are measured before the insulin or glucose challenge and at 15, 20 or 30 minute intervals for up to 3 hours.

To assess the metabolic rate of diet-induced obese mice treated with the oligomeric compounds of the invention, the respiratory quotient and oxygen consumption of the mice is also measured.

The diet-induced obese mice that receive treatment are evaluated at the end of the treatment period for the effects of target modulation on the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism, as described supra.

P-407 Mouse Model of Hyperlipidemia:

Poloxamer 407 (P-407), an inert block copolymer comprising a hydrophobic core flanked by hydrophilic polyoxyethelene units has been shown to induce hyperlipidemia in rodents. In the mouse, one injection, intraperitoneally, of P-407 (0.5 g/kg) produced hypercholesterolemia that peaked at 24 hours and returned to control levels by 96 hours following treatment (Palmer, et al., Atherosclerosis, 1998, 136, 115-123). Consequently, these mice are a useful model for the investigation of compounds that modulate hyperlipidemia. In accordance with the present invention, the oligomeric compounds of the invention are tested in the P-407 model of hyperlipidemia.

Seven-week old male C57B1/6 mice are divided into two groups; (1) control and (2) P-407 injected animals (0.5 g/kg every 3 days, following an overnight fast). Animals in each group receive either a saline injection or injection with one or more of the oligomeric compounds of the invention or control compounds at 25 mg/kg three times per week or 50 mg/kg two times per week. All injections are administered intraperitoneally.

After the treatment period, mice are sacrificed and target levels are evaluated in liver, BAT and WAT as described supra.

To assess the physiological effects resulting from modulation of target, the P-407 injected animals that receive treatment are evaluated at the end of the treatment period for serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis is assessed by measuring the liver triglyceride content and oil red O staining, as described supra.

The effects of target modulation on glucose and insulin metabolism are evaluated in the P-407 injected animals treated with the oligomeric compounds of the invention. Plasma glucose is measured at the start of the treatment and after 2 weeks and 4 weeks of treatment. Plasma insulin is similarly measured at the beginning of the treatment, and following 2 weeks and 4 weeks of treatment. Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose and insulin levels are measured before the insulin or glucose challenge and at 15, 20 or 30 minute intervals for up to 3 hours.

To assess the metabolic rate of P-407 injected animals treated with the oligomeric compounds of the invention, the respiratory quotient and oxygen consumption of the mice is measured.

The P-407 injected animals that receive treatment are evaluated at the end of the treatment period for the effects of target modulation on the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism, as described supra.

ApoE Knockout Mice (A Model of Dyslipidemia and Obesity):

B6.129P-ApoE^(tm1Unc) knockout mice (herein referred to as ApoE knockout mice) obtained from The Jackson Laboratory (Bar Harbor, Me.), are homozygous for the Apoe^(tm1Unc) mutation and show a marked increase in total plasma cholesterol levels that are unaffected by age or sex. These animals present with fatty streaks in the proximal aorta at 3 months of age. These lesions increase with age and progress to lesions with less lipid but more elongated cells, typical of a more advanced stage of pre-atherosclerotic lesion.

The mutation in these mice resides in the apolipoprotein E (ApoE) gene. The primary role of the ApoE protein is to transport cholesterol and triglycerides throughout the body. It stabilizes lipoprotein structure, binds to the low density lipoprotein receptor (LDLR) and related proteins, and is present in a subclass of HDLs, providing them the ability to bind to LDLR. ApoE is expressed most abundantly in the liver and brain. In one embodiment of the present invention, female B6.129P-Apoetm1Unc knockout mice (ApoE knockout mice) are used in the following studies to evaluate the oligomeric compounds of the invention as potential lipid lowering compounds.

Female ApoE knockout mice range in age from 5 to 7 weeks and are placed on a normal diet for 2 weeks before study initiation. ApoE knockout mice are then fed ad libitum a 60% fat diet, with 0.15% added cholesterol to induce dyslipidemia and obesity. Control animals include ApoE knockout mice and ApoE wildtype mice (i.e. lean littermates) maintained on a high-fat diet with no added cholesterol. After overnight fasting, mice from each group are dosed intraperitoneally every three days with saline, 50 mg/kg of a control compound or 5, 25 or 50 mg/kg of one or more of the oligomeric compounds of the invention.

After the treatment period, mice are sacrificed and target levels are evaluated in liver, BAT and WAT as described supra.

To assess the physiological effects resulting from modulation of target, the ApoE knockout mice that receive treatment are evaluated at the end of the treatment period for serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis is assessed by measuring the liver triglyceride content and oil red O staining, as described supra.

The effects of target modulation on glucose and insulin metabolism are also evaluated in the ApoE knockout mice treated with the oligomeric compounds of the invention. Plasma glucose is measured at the start of the treatment and after 2 weeks and 4 weeks of treatment. Plasma insulin is similarly measured at the beginning of the treatment, and following 2 weeks and 4 weeks of treatment. Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose and insulin levels are measured before the insulin or glucose challenge and at 15, 20 or 30 minute intervals for up to 3 hours.

To assess the metabolic rate of ApoE knockout mice treated with the oligomeric compounds of the invention, the respiratory quotient and oxygen consumption of the mice are measured.

The ApoE knockout mice that receive treatment are evaluated at the end of the treatment period for the effects of target modulation on the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism, as described supra.

In order that the invention disclosed herein may be more efficiently understood, examples are provided below. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the invention in any manner. Throughout these examples, molecular cloning reactions, and other standard recombinant DNA techniques, were carried out according to methods described in Maniatis et al., Molecular Cloning—A Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989), using commercially available reagents, except where otherwise noted.

EXAMPLES Example 1 Oligomeric Compounds Targeting Small Non-Coding RNAs

In accordance with the present invention, a series of oligomeric compounds are designed to target different regions of small non-coding target RNAs. The oligomeric compounds can be investigated for their effect on small non-coding RNA levels by quantitative real-time PCR. The target regions to which these sequences are complementary are herein referred to as “suitable target regions”.

Example 2 Oligomeric Compounds that Mimic or Replace Small Non-Coding RNAs

In accordance with the present invention, a series of oligomeric compounds are designed to mimic the structure and/or function of small non-coding RNAs. These mimics may include isolated single-, double-, or multiple-stranded compounds, any of which may include regions of intrastrand nucleobase complementarity, said regions capable of folding and forming a molecule with fully or partially double-stranded or multiple-stranded character based on regions of precise or imperfect complementarity. The oligomeric compound mimics can then be investigated for their effects on a cell, tissue or organism system lacking endogenous small non-coding RNAs or systems with aberrant expression of small non-coding RNAs using the screening methods disclosed herein or those commonly used in the art. Changes in levels, expression or function of the small non-coding RNA or its downstream target nucleic acid levels can be analyzed by quantitative real-time PCR as described, supra.

Example 3 Pri-miRNAs Targeted by Compounds of the Present Invention

In accordance with the present invention, oligomeric compounds were designed to target one or more microRNA (miRNA) genes or gene products. Certain pri-miRNAs have been reported by Lim et al. Science, 2003, 299; 1540; in Brevia (detailed in the supplemental online materials; www.sciencemag.org/cgi/content/full/299/5612/1540/DC1) and these were used as starting targets.

A list of pri-miRNAs targeted is shown in Table 1. The gene name for each of the 188 targets (assigned by Lim et al.) is given in the table. For those pri-miRNAs that did not produce an identifiable miRNA detectable by PCR in the Lim publication, the position and sequence of the miRNAs were identified herein and are referred to as novel or hypothetical miRNAs. Also shown is the Genbank Accession number of the source sequence from which the pri-miRNA was extracted. The sequence is set forth in the Sequence Listing and is written in the 5′ to 3′ direction and is represented in the DNA form. It is understood that a person having ordinary skill in the art would be able to convert the sequence of the targets to their RNA form by simply replacing the thymidine (T) with uracil (U) in the sequence.

TABLE 1 pri-miRNAs Genbank Accession # of source SEQ pri-miRNA sequence ID NO mir-140 NT_037896.1 4 mir-30a NT_007299.11 5 mir-34 NT_028054.10 6 mir-29b-1 NT_021877.13 7 mir-29b-2 NT_007933.10 8 mir-16-3 NT_005612.11 9 mir-203 NT_026437.9 10 mir-7-1 NT_023935.13 11 mir-10b NT_037537.1 12 mir-128a NT_034487.2 13 mir-153-1 NT_005403.11 14 mir-153-2 NT_007741.10 15 hypothetical miRNA-013 NT_010194.13 16 mir-27b NT_008476.13 17 mir-96 NT_007933.10 18 mir-17as/mir-91 NT_009952.11 19 mir-123/mir-126as NT_024000.13 20 mir-132 NT_010692.9 21 mir-108-1 NT_010799.11 22 mir-23b NT_008476.13 23 let-7i NT_009711.13 24 mir-212 NT_010692.9 25 hypothetical miRNA-023 NT_004658.12 26 mir-131-2 NT_029973.6 27 let-7b NT_011523.8 28 mir-1d NT_035608.1 29 mir-122a NT_033907.3 30 mir-22 NT_010692.9 31 mir-92-1 NT_009952.11 32 hypothetical miRNA-030 NT_007933.10 33 mir-142 NT_010783.11 34 mir-183 NT_007933.10 35 hypothetical miRNA-033 NT_011588.11 36 mir-214 NT_029874.7 37 mir-143 NT_006859.11 38 mir-192-1 NT_033241.3 39 mir-192-2 NT_033241.3 39 mir-192-3 NT_033241.3 39 hypothetical miRNA-039 NT_028392.4 42 hypothetical miRNA-040 NT_023148.9 43 hypothetical miRNA-041 NT_023089.11 44 let-7a-3 NT_011523.8 45 hypothetical miRNA-043 NT_004902.12 46 hypothetical miRNA-044 NT_009952.11 47 mir-181a NT_017568.11 48 let-7a-1 NT_008476.13 49 mir-205 NT_021877.13 50 mir-103-1 NT_037665.1 51 mir-26a NT_005580.13 52 mir-33a NT_011520.8 53 mir-196-2 NT_009458.12 54 mir-107 NT_033890.3 55 mir-106 NT_011786.11 56 let-7f-1 NT_008476.13 57 hypothetical miRNA-055 NT_006713.11 58 mir-29c NT_021877.13 59 mir-130a NT_033903.3 60 hypothetical miRNA-058 NT_037537.1 61 mir-218-1 NT_006316.13 62 mir-124a-2 NT_008183.13 63 mir-21 NT_035426.2 64 mir-16-1 NT_033922.3 65 mir-144 NT_010799.11 66 mir-221 NT_011568.10 67 mir-222 NT_011568.10 68 mir-30d NT_028251.8 69 mir-19b-2 NT_011786.11 70 mir-128b NT_005580.13 71 hypothetical miRNA-069 NT_017568.11 72 hypothetical miRNA-070 NT_005375.11 73 hypothetical miRNA-071 NT_011512.7 74 mir-29b-3 NT_007933.10 75 mir-129-2 NT_009237.13 76 mir-133b NT_007592.11 77 hypothetical miRNA-075 NT_006044.8 78 let-7d NT_008476.13 79 mir-15b NT_005612.11 80 mir-29a-1 NT_007933.10 81 hypothetical miRNA-079 NT_021907.13 82 mir-199b NT_017568.11 83 mir-129-1 NT_007933.10 84 let-7e NT_011109.13 85 hypothetical miRNA-083 NT_024524.11 86 let-7c NT_011512.7 87 mir-204 NT_008580.11 88 mir-145 NT_006859.11 89 mir-124a-1 NT_019483.13 90 hypothetical miRNA-088 NT_011519.9 91 mir-213 NT_029862.8 92 hypothetical miRNA-090 NT_006171.13 93 mir-20 NT_009952.11 94 mir-133a-1 NT_011044.11 95 mir-138-2 NT_010498.11 96 mir-98 NT_011799.10 97 mir-196-1 NT_010783.11 98 mir-125b-1 NT_033899.3 99 mir-199a-2 NT_029874.7 100 mir-29a-2 NT_007933.10 101 hypothetical miRNA-099 NT_016297.12 102 mir-181b NT_029862.8 103 hypothetical miRNA-101 NT_030828.7 104 mir-141 NT_035206.1 105 mir-131-1 NT_004858.13 106 mir-133a-2 NT_035608.1 107 hypothetical miRNA-105 NT_017795.13 108 hypothetical miRNA-106 NT_017795.13 109 hypothetical miRNA-107 NT_008583.13 110 mir-1b NT_011044.11 111 mir-18 NT_009952.11 112 mir-220 NT_011588.11 113 hypothetical miRNA-111 NT_004525.13 114 mir-7-3 NT_011255.11 115 mir-218-2 NT_023132.10 116 mir-24-2 NT_031915.4 117 mir-24-1 NT_008476.13 118 mir-103-2 NT_011387.8 119 mir-211 NT_010363.13 120 mir-101-3 NT_008413.13 121 mir-30b NT_028251.8 122 hypothetical miRNA-120 NT_009952.11 123 let-7a-4 NT_033899.3 124 mir-10a NT_010783.11 125 mir-19a NT_009952.11 126 let-7f-2 NT_011799.10 127 mir-15a-1 NT_010393.11 128 mir-108-2 NT_034392.2 129 mir-137 NT_033951.3 130 mir-219 NT_007592.11 131 mir-148b NT_009458.12 132 mir-130b NT_011520.8 133 mir-19b-1 NT_009952.11 134 let-7a-2 NT_033899.3 135 mir-216 NT_005375.11 136 mir-100-1 NT_033899.3 137 mir-100-2 NT_033899.3 137 mir-187 NT_010966.11 139 hypothetical miRNA-137 NT_011387.8 140 hypothetical miRNA-138 NT_008902.13 141 hypothetical miRNA-139 NT_008902.13 142 mir-124a-3 NT_011333.5 143 mir-7-2 NT_033276.3 144 hypothetical miRNA-142 NT_033317.3 145 hypothetical miRNA-143 NT_007819.11 146 hypothetical miRNA-144 NT_010783.11 147 mir-210 NT_035113.2 148 mir-215 NT_021953.13 149 mir-223 NT_011669.11 150 mir-131-3 NT_033276.3 151 mir-199a-1 NT_011176.13 152 mir-30c NT_007299.11 153 mir-101-1 NT_029865.8 154 mir-101-2 NT_029865.8 154 hypothetical miRNA-153 NT_005332.11 156 hypothetical miRNA-154 NT_030828.7 157 mir-26b NT_005403.11 158 hypothetical miRNA-156 NT_029289.7 159 mir-152 NT_010783.11 160 mir-135-1 NT_005986.13 161 mir-135-2 NT_009681.13 162 mir-217 NT_005375.11 163 hypothetical miRNA-161 NT_004658.12 164 mir-15a-2 NT_033922.3 165 let-7g NT_005986.13 166 hypothetical miRNA-164 NT_010783.11 167 mir-33b NT_030843.4 168 hypothetical miRNA-166 NT_011588.11 169 mir-16-2 NT_033922.3 170 hypothetical miRNA-168 NT_011520.8 171 hypothetical miRNA-169 NT_007933.10 172 hypothetical miRNA-170 NT_005151.11 173 hypothetical miRNA-171 NT_006171.13 174 hypothetical miRNA-172 NT_037752.1 175 hypothetical miRNA-173 NT_008413.13 176 mir-182 NT_007933.10 177 hypothetical miRNA-175 NT_006258.12 178 hypothetical miRNA-176 NT_025004.11 179 hypothetical miRNA-177 NT_023098.7 180 hypothetical miRNA-178 NT_037537.1 181 hypothetical miRNA-179 NT_010194.13 182 hypothetical miRNA-180 NT_010363.13 183 hypothetical miRNA-181 NT_033899.3 184 mir-148a NT_007819.11 185 hypothetical miRNA-183 NT_010363.13 186 mir-23a NT_031915.4 187 hypothetical miRNA-185 NT_007592.11 188 hypothetical miRNA-186 NT_008705.13 189 mir-181c NT_031915.4 190 hypothetical miRNA-188 NT_023148.9 191

Example 4 miRNAs within Pri-miRNAs

miRNAs found within the pri-miRNA structures disclosed above were used in certain embodiments of the present invention. These miRNAs represent target nucleic acids to which the oligomeric compounds of the present invention were designed. The oligomeric compounds of the present invention can also be designed to mimic the miRNA while incorporating certain chemical modifications that alter one or more properties of the mimic, thereby creating a construct with superior properties over the endogenous miRNA. The miRNA target sequences are shown in Table 2.

TABLE 2  miRNAs found within pri-miRNAs miRNA sequence SEQ  DNA form; where T ID Pri-miRNA (replaces U in RNA) NO mir-140 AGTGGTTTTACCCTATGGTAG 192 mir-30a CTTTCAGTCGGATGTTTGCAGC 193 mir-34 TGGCAGTGTCTTAGCTGGTTGT 194 mir-29b-1 TAGCACCATTTGAAATCAGTGTT 195 mir-29b-2 TAGCACCATTTGAAATCAGTGTT 195 mir-16-3 TAGCAGCACGTAAATATTGGCG 196 mir-203 GTGAAATGTTTAGGACCACTAG 197 mir-7-1 TGGAAGACTAGTGATTTTGTT 198 mir-10b TACCCTGTAGAACCGAATTTGT 199 mir-128a TCACAGTGAACCGGTCTCTTTT 200 mir-153-1 TTGCATAGTCACAAAAGTGA 201 mir-153-2 TTGCATAGTCACAAAAGTGA 201 mir-27b TTCACAGTGGCTAAGTTCTG 202 mir-96 TTTGGCACTAGCACATTTTTGC 203 mir-l7as/mir-91 CAAAGTGCTTACAGTGCAGGTA 204 GT mir-123/mir-126as CATTATTACTTTTGGTACGCG 205 mir-132 TAACAGTCTACAGCCATGGTCGC 206 mir-108-1 ATAAGGATTTTTAGGGGCATT 207 mir-23b ATCACATTGCCAGGGATTACCAC 208 let-7i TGAGGTAGTAGTTTGTGCT 209 mir-212 TAACAGTCTCCAGTCACGGCC 210 mir-131-2 TAAAGCTAGATAACCGAAAGT 211 let-7b TGAGGTAGTAGGTTGTGTGGTT 212 mir-1d TGGAATGTAAAGAAGTATGTAT 213 mir-122a TGGAGTGTGACAATGGTGTTTGT 214 mir-22 AAGCTGCCAGTTGAAGAACTGT 215 mir-92-1 TATTGCACTTGTCCCGGCCTGT 216 mir-142 CATAAAGTAGAAAGCACTAC 217 mir-183 TATGGCACTGGTAGAATTCACTG 218 mir-214 ACAGCAGGCACAGACAGGCAG 219 mir-143 TGAGATGAAGCACTGTAGCTCA 220 mir-192-1 CTGACCTATGAATTGACAGCC 221 mir-192-2 CTGACCTATGAATTGACAGCC 221 mir-192-3 CTGACCTATGAATTGACAGCC 221 let-7a-3 TGAGGTAGTAGGTTGTATAGTT 222 mir-181a AACATTCAACGCTGTCGGTGAGT 223 let-7a-1 TGAGGTAGTAGGTTGTATAGTT 222 mir-205 TCCTTCATTCCACCGGAGTCTG 224 mir-103-1 AGCAGCATTGTACAGGGCTATGA 225 mir-26a TTCAAGTAATCCAGGATAGGCT 226 mir-33a GTGCATTGTAGTTGCATTG 227 mir-196-2 TAGGTAGTTTCATGTTGTTGGG 228 mir-107 AGCAGCATTGTACAGGGCTATCA 229 mir-106 AAAAGTGCTTACAGTGCAGGTAGC 230 let-7f-1 TGAGGTAGTAGATTGTATAGTT 231 mir-29c CTAGCACCATTTGAAATCGGTT 232 mir-130a CAGTGCAATGTTAAAAGGGC 233 mir-218-1 TTGTGCTTGATCTAACCATGT 234 mir-124a-2 TTAAGGCACGCGGTGAATGCCA 235 mir-21 TAGCTTATCAGACTGATGTTGA 236 mir-16-1 TAGCAGCACGTAAATATTGGCG 196 mir-144 TACAGTATAGATGATGTACTAG 237 mir-221 AGCTACATTGTCTGCTGGGTTTC 238 mir-222 AGCTACATCTGGCTACTGGGTCTC 239 mir-30d TGTAAACATCCCCGACTGGAAG 240 mir-19b-2 TGTGCAAATCCATGCAAAACTGA 241 mir-128b TCACAGTGAACCGGTCTCTTTC 242 mir-29b-3 TAGCACCATTTGAAATCAGTGTT 195 mir-129-2 CTTTTTGCGGTCTGGGCTTGC 243 mir-133b TTGGTCCCCTTCAACCAGCTA 244 let-7d AGAGGTAGTAGGTTGCATAGT 245 mir-15b TAGCAGCACATCATGGTTTACA 246 mir-29a-1 CTAGCACCATCTGAAATCGGTT 247 mir-199b CCCAGTGTTTAGACTATCTGTTC 248 mir-129-1 CTTTTTGCGGTCTGGGCTTGC 243 let-7e TGAGGTAGGAGGTTGTATAGT 249 let-7c TGAGGTAGTAGGTTGTATGGTT 250 mir-204 TTCCCTTTGTCATCCTATGCCT 251 mir-145 GTCCAGTTTTCCCAGGAATCCC 252 TT mir-124a-1 TTAAGGCACGCGGTGAATGCCA 235 mir-213 ACCATCGACCGTTGATTGTACC 253 mir-20 TAAAGTGCTTATAGTGCAGGTAG 254 mir-133a-1 TTGGTCCCCTTCAACCAGCTGT 255 mir-138-2 AGCTGGTGTTGTGAATC 256 mir-98 TGAGGTAGTAAGTTGTATTGTT 257 mir-196-1 TAGGTAGTTTCATGTTGTTGGG 228 mir-125b-1 TCCCTGAGACCCTAACTTGTGA 258 mir-199a-2 CCCAGTGTTCAGACTACCTGTTC 259 mir-29a-2 CTAGCACCATCTGAAATCGGTT 247 mir-181b AACATTCATTGCTGTCGGTGGG 260 TT mir-141 AACACTGTCTGGTAAAGATGG 261 mir-131-1 TAAAGCTAGATAACCGAAAGT 211 mir-133a-2 TTGGTCCCCTTCAACCAGCTGT 255 mir-1b TGGAATGTAAAGAAGTATGTAT 213 mir-18 TAAGGTGCATCTAGTGCAGATA 262 mir-220 CCACACCGTATCTGACACTTT 263 mir-7-3 TGGAAGACTAGTGATTTTGTT 198 mir-218-2 TTGTGCTTGATCTAACCATGT 234 mir-24-2 TGGCTCAGTTCAGCAGGAACAG 264 mir-24-1 TGGCTCAGTTCAGCAGGAACAG 264 mir-103-2 AGCAGCATTGTACAGGGCTATGA 225 mir-211 TTCCCTTTGTCATCCTTCGCCT 264 mir-101-3 TACAGTACTGTGATAACTGA 265 mir-30b TGTAAACATCCTACACTCAGC 266 let-7a-4 TGAGGTAGTAGGTTGTATAGTT 222 mir-10a TACCCTGTAGATCCGAATTTGTG 267 mir-19a TGTGCAAATCTATGCAAAACTGA 268 let-7f-2 TGAGGTAGTAGATTGTATAGTT 231 mir-15a-1 TAGCAGCACATAATGGTTTGTG 269 mir-108-2 ATAAGGATTTTTAGGGGCATT 207 mir-137 TATTGCTTAAGAATACGCGTAG 270 mir-219 TGATTGTCCAAACGCAATTCT 271 mir-148b TCAGTGCATCACAGAACTTTGT 272 mir-130b CAGTGCAATGATGAAAGGGC 273 mir-19b-1 TGTGCAAATCCATGCAAAACTGA 241 let-7a-2 TGAGGTAGTAGGTTGTATAGTT 222 mir-216 TAATCTCAGCTGGCAACTGTG 274 mir-100-1 AACCCGTAGATCCGAACTTGTG 275 mir-100-2 AACCCGTAGATCCGAACTTGTG 275 mir-187 TCGTGTCTTGTGTTGCAGCCGG 276 mir-124a-3 TTAAGGCACGCGGTGAATGCCA 235 mir-7-2 TGGAAGACTAGTGATTTTGTT 198 mir-210 CTGTGCGTGTGACAGCGGCTG 277 mir-215 ATGACCTATGAATTGACAGAC 278 mir-223 TGTCAGTTTGTCAAATACCCC 279 mir-131-3 TAAAGCTAGATAACCGAAAGT 211 mir-199a-1 CCCAGTGTTCAGACTACCTGTTC 259 mir-30c TGTAAACATCCTACACTCTCAGC 280 mir-101-1 TACAGTACTGTGATAACTGA 265 mir-101-2 TACAGTACTGTGATAACTGA 265 mir-26b TTCAAGTAATTCAGGATAGGTT 281 mir-152 TCAGTGCATGACAGAACTTGG 282 mir-135-1 TATGGCTTTTTATTCCTATGTG 283 AT mir-135-2 TATGGCTTTTTATTCCTATGTG 283 AT mir-217 TACTGCATCAGGAACTGATTGG 284 AT mir-15a-2 TAGCAGCACATAATGGTTTGTG 269 let-7g TGAGGTAGTAGTTTGTACAGT 285 mir-33b GTGCATTGCTGTTGCATTG 286 mir-16-2 TAGCAGCACGTAAATATTGGCG 196 mir-182 TTTGGCAATGGTAGAACTCACA 287 mir-148a TCAGTGCACTACAGAACTTTGT 288 mir-23a ATCACATTGCCAGGGATTTCC 289 mir-181c AACATTCAACCTGTCGGTGAGT 290

Example 5 Uniform 2′-MOE Phosphorothioate (PS) Oligomeric Compounds Targeting miRNAs

In accordance with the present invention, a series of oligomeric compounds were designed and synthesized to target miRNA sequences disclosed by Lim et al. Science, 2003, 299, 1540. The compounds are shown in Table 3. “Pri-miRNA” indicates the particular pri-miRNA which contains the miRNA that the oligomeric compound was designed to target. All compounds in Table 3 are composed of 2′-methoxyethoxy (2′-MOE) nucleotides throughout and the internucleoside (backbone) linkages are phosphorothioate (P═S) throughout. All cytidine residues are 5-methylcytidines. The compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR as described, supra, or they can be used in other assays to investigate the role of miRNAs or the function of targets downstream of miRNAs.

TABLE 3 Uniform 2′-MOE PS Compounds targeting miRNAs SEQ ISIS ID  Number NO  Sequence Pri-miRNA 327873 291 CTACCATAGGGTAAAACCACT mir-140 327874 292 GCTGCAAACATCCGACTGAAAG mir-30a 327875 293 ACAACCAGCTAAGACACTGCCA mir-34 327876 294 AACACTGATTTCAAATGGTGCTA mir-29b-1 327877 295 CGCCAATATTTACGTGCTGCTA mir-16-3 327878 296 CTAGTGGTCCTAAACATTTCAC mir-203 327879 297 AACAAAATCACTAGTCTTCCA mir-7-1 327880 298 ACAAATTCGGTTCTACAGGGTA mir-10b 327881 299 AAAAGAGACCGGTTCACTGTGA mir-128a 327882 300 TCACTTTTGTGACTATGCAA mir-153-1 327883 301 CAGAACTTAGCCACTGTGAA mir-27b 327884 302 GCAAAAATGTGCTAGTGCCAAA mir-96 327885 303 ACTACCTGCACTGTAAGCACTTTG mir-l7as/ mir-91 327886 304 CGCGTACCAAAAGTAATAATG mir-123/ mir-126as 327887 305 GCGACCATGGCTGTAGACTGTTA mir-132 327888 306 AATGCCCCTAAAAATCCTTAT mir-108-1 327889 307 GTGGTAATCCCTGGCAATGTGAT mir-23b 327890 308 AGCACAAACTACTACCTCA let-7i 327891 309 GGCCGTGACTGGAGACTGTTA mir-212 327892 310 ACTTTCGGTTATCTAGCTTTA mir-131-2/ mir-9 327893 311 AACCACACAACCTACTACCTCA let-7b 327894 312 ATACATACTTCTTTACATTCCA mir-1d 327895 313 ACAAACACCATTGTCACACTCCA mir-122a 327896 314 ACAGTTCTTCAACTGGCAGCTT mir-22 327897 315 ACAGGCCGGGACAAGTGCAATA mir-92-1 327898 316 GTAGTGCTTTCTACTTTATG mir-142 327899 317 CAGTGAATTCTACCAGTGCCATA mir-183 327900 318 CTGCCTGTCTGTGCCTGCTGT mir-214 327901 319 TGAGCTACAGTGCTTCATCTCA mir-143 327902 320 GGCTGTCAATTCATAGGTCAG mir-192-1 327903 321 AACTATACAACCTACTACCTCA let-7a-3 327904 322 ACTCACCGACAGCGTTGAATGTT mir-181a 327905 323 CAGACTCCGGTGGAATGAAGGA mir-205 327906 324 TCATAGCCCTGTACAATGCTGCT mir-103-1 327907 325 AGCCTATCCTGGATTACTTGAA mir-26a 327908 326 CAATGCAACTACAATGCAC mir-33a 327909 327 CCCAACAACATGAAACTACCTA mir-196-2 327910 328 TGATAGCCCTGTACAATGCTGCT mir-107 327911 329 GCTACCTGCACTGTAAGCACTTTT mir-106 327912 330 AACTATACAATCTACTACCTCA let-7f-1 327913 331 AACCGATTTCAAATGGTGCTAG mir-29c 327914 332 GCCCTTTTAACATTGCACTG mir-130a 327915 333 ACATGGTTAGATCAAGCACAA mir-218-1 327916 334 TGGCATTCACCGCGTGCCTTAA mir-124a-2 327917 335 TCAACATCAGTCTGATAAGCTA mir-21 327918 336 CTAGTACATCATCTATACTGTA mir-144 327919 337 GAAACCCAGCAGACAATGTAGCT mir-221 327920 338 GAGACCCAGTAGCCAGATGTAGCT mir-222 327921 339 CTTCCAGTCGGGGATGTTTACA mir-30d 327922 340 TCAGTTTTGCATGGATTTGCACA mir-19b-2 327923 341 GAAAGAGACCGGTTCACTGTGA mir-128b 327924 342 GCAAGCCCAGACCGCAAAAAG mir-129-2 327925 343 TAGCTGGTTGAAGGGGACCAA mir-133b 327926 344 ACTATGCAACCTACTACCTCT let-7d 327927 345 TGTAAACCATGATGTGCTGCTA mir-15b 327928 346 AACCGATTTCAGATGGTGCTAG mir-29a-1 327929 347 GAACAGATAGTCTAAACACTGGG mir-199b 327930 348 ACTATACAACCTCCTACCTCA let-7e 327931 349 AACCATACAACCTACTACCTCA let-7c 327932 350 AGGCATAGGATGACAAAGGGAA mir-204 327933 351 AAGGGATTCCTGGGAAAACTGGAC mir-145 327934 352 GGTACAATCAACGGTCGATGGT mir-213 327935 353 CTACCTGCACTATAAGCACTTTA mir-20 327936 354 ACAGCTGGTTGAAGGGGACCAA mir-133a-1 327937 355 GATTCACAACACCAGCT mir-138-2 327938 356 AACAATACAACTTACTACCTCA mir-98 327939 357 TCACAAGTTAGGGTCTCAGGGA mir-125b-1 327940 358 GAACAGGTAGTCTGAACACTGGG mir-199a-2 327941 359 AACCCACCGACAGCAATGAATGTT mir-181b 327942 360 CCATCTTTACCAGACAGTGTT mir-141 327943 361 TATCTGCACTAGATGCACCTTA mir-18 327944 362 AAAGTGTCAGATACGGTGTGG mir-220 327945 363 CTGTTCCTGCTGAACTGAGCCA mir-24-2 327946 364 AGGCGAAGGATGACAAAGGGAA mir-211 327947 365 TCAGTTATCACAGTACTGTA mir-101-3 327948 366 GCTGAGTGTAGGATGTTTACA mir-30b 327949 367 CACAAATTCGGATCTACAGGGTA mir-10a 327950 368 TCAGTTTTGCATAGATTTGCACA mir-19a 327951 369 CACAAACCATTATGTGCTGCTA mir-15a-1 327952 370 CTACGCGTATTCTTAAGCAATA mir-137 327953 371 AGAATTGCGTTTGGACAATCA mir-219 327954 372 ACAAAGTTCTGTGATGCACTGA mir-148b 327955 373 GCCCTTTCATCATTGCACTG mir-130b 327956 374 CACAGTTGCCAGCTGAGATTA mir-216 327957 375 CACAAGTTCGGATCTACGGGTT mir-100-1 327958 376 CCGGCTGCAACACAAGACACGA mir-187 327959 377 CAGCCGCTGTCACACGCACAG mir-210 327960 378 GTCTGTCAATTCATAGGTCAT mir-215 327961 379 GGGGTATTTGACAAACTGACA mir-223 327962 380 GCTGAGAGTGTAGGATGTTTACA mir-30c 327963 381 AACCTATCCTGAATTACTTGAA mir-26b 327964 382 CCAAGTTCTGTCATGCACTGA mir-152 327965 383 ATCACATAGGAATAAAAAGCCATA mir-135-1 327966 384 ATCCAATCAGTTCCTGATGCAGTA mir-217 327967 385 ACTGTACAAACTACTACCTCA let-7g 327968 386 CAATGCAACAGCAATGCAC mir-33b 327969 387 TGTGAGTTCTACCATTGCCAAA mir-182 327970 388 ACAAAGTTCTGTAGTGCACTGA mir-148a 327971 389 GGAAATCCCTGGCAATGTGAT mir-23a 327972 390 ACTCACCGACAGGTTGAATGTT mir-181c

Example 6 Uniform 2′-MOE Phosphorothioate (PS) Oligomeric Compounds Targeting Novel miRNAs

In accordance with the present invention, a series of oligomeric compounds were designed and synthesized to target novel miRNAs. The compounds are shown in Table 4. “Pri-miRNA” indicates the particular pri-miRNA defined herein which contains the miRNA that the oligomeric compound was designed to target. The sequence of the compounds represent the full complement of the novel miRNA defined herein. All compounds in Table 4 are composed of 2′-methoxyethoxy (2′-MOE) nucleotides throughout and the internucleoside (backbone) linkages are phosphorothioate (P═S) throughout. All cytidine residues are 5-methylcytidines. The compounds can be analyzed for their effect miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR as described, supra, or they can be used in other assays to investigate the role of miRNAs or downstream targets of miRNAs.

TABLE 4 Uniform 2′-MOE PS Compounds  targeting novel pri-miRNAs SEQ  ISIS ID Sequence Number NO (5′-3′) Pri-miRNA 328089 391 ACTGTAGGAATATGTTTGATA hypothetical  miRNA-013 328090 392 ATTAAAAAGTCCTCTTGCCCA hypothetical  miRNA-023 328091 393 GCTGCCGTATATGTGATGTCA hypothetical  miRNA-030 328092 394 GGTAGGTGGAATACTATAACA hypothetical  miRNA-033 328093 395 TAAACATCACTGCAAGTCTTA hypothetical  miRNA-039 328094 396 TTGTAAGCAGTTTTGTTGACA hypothetical  miRNA-040 328095 397 TCACAGAGAAAACAACTGGTA hypothetical  miRNA-041 328096 398 CCTCTCAAAGATTTCCTGTCA hypothetical  miRNA-043 328097 399 TGTCAGATAAACAGAGTGGAA hypothetical  miRNA-044 328098 400 GAGAATCAATAGGGCATGCAA hypothetical  miRNA-055 328099 401 AAGAACATTAAGCATCTGACA hypothetical  miRNA-058 328100 402 AATCTCTGCAGGCAAATGTGA hypothetical  miRNA-070 328101 403 AAACCCCTATCACGATTAGCA hypothetical  miRNA-071 328102 404 GCCCCATTAATATTTTAACCA hypothetical  miRNA-075 328103 405 CCCAATATCAAACATATCA hypothetical  miRNA-079 328104 406 TATGATAGCTTCCCCATGTAA hypothetical  miRNA-083 328105 407 CCTCAATTATTGGAAATCACA hypothetical  miRNA-088 328106 408 ATTGATGCGCCATTTGGCCTA hypothetical  miRNA-090 328107 409 CTGTGACTTCTCTATCTGCCT hypothetical  miRNA-099 328108 410 AAACTTGTTAATTGACTGTCA hypothetical  miRNA-l0l 328109 411 AAAGAAGTATATGCATAGGAA hypothetical  miRNA-l05 328110 412 GATAAAGCCAATAAACTGTCA hypothetical  miRNA-107 328111 413 TCCGAGTCGGAGGAGGAGGAA hypothetical  miRNA-111 328112 414 ATCATTACTGGATTGCTGTAA hypothetical  miRNA-120 328113 415 CAAAAATTATCAGCCAGTTTA hypothetical  miRNA-137 328114 416 AATCTCATTTTCATACTTGCA hypothetical  miRNA-138 328115 417 AGAAGGTGGGGAGCAGCGTCA hypothetical  miRNA-142 328116 418 CAAAATTGCAAGCAAATTGCA hypothetical  miRNA-143 328117 419 TCCACAAAGCTGAACATGTCT hypothetical  miRNA-144 328118 420 TATTATCAGCATCTGCTTGCA hypothetical  miRNA-153 328119 421 AATAACACACATCCACTTTAA hypothetical  miRNA-154 328120 422 AAGAAGGAAGGAGGGAAAGCA hypothetical  miRNA-156 328121 423 ATGACTACAAGTTTATGGCCA hypothetical  miRNA-161 328122 424 CAAAACATAAAAATCCTTGCA hypothetical  miRNA-164 328123 425 TTACAGGTGCTGCAACTGGAA hypothetical  miRNA-166 328124 426 AGCAGGTGAAGGCACCTGGCT hypothetical  miRNA-168 328125 427 TATGAAATGCCAGAGCTGCCA hypothetical  miRNA-169 328126 428 CCAAGTGTTAGAGCAAGATCA hypothetical  miRNA-170 328127 429 AACGATAAAACATACTTGTCA hypothetical  miRNA-171 328128 430 AGTAACTTCTTGCAGTTGGA hypothetical  miRNA-172 328129 431 AGCCTCCTTCTTCTCGTACTA hypothetical  miRNA-173 328130 432 ACCTCAGGTGGTTGAAGGAGA hypothetical  miRNA-175 328131 433 ATATGTCATATCAAACTCCTA hypothetical  miRNA-176 328132 434 GTGAGAGTAGCATGTTTGTCT hypothetical  miRNA-177 328133 435 TGAAGGTTCGGAGATAGGCTA hypothetical  miRNA-178 328134 436 AATTGGACAAAGTGCCTTTCA hypothetical miRNA-179 328135 437 ACCGAACAAAGTCTGACAGGA hypothetical  miRNA-180 328136 438 AACTACTTCCAGAGCAGGTGA hypothetical  miRNA-181 328137 439 GTAAGCGCAGCTCCACAGGCT hypothetical  miRNA-183 328138 440 GAGCTGCTCAGCTGGCCATCA hypothetical  miRNA-185 328139 441 TACTTTTCATTCCCCTCACCA hypothetical  miRNA-188

Example 7 Chimeric Phosphorothioate Compounds Having 2′-MOE Wings and a Deoxy Gap Targeting Pri-miRNAs

In accordance with the present invention, a series of oligomeric compounds were designed and synthesized to target different regions of pri-miRNA structures. The compounds are shown in Table 5. “Pri-miRNA” indicates the particular pri-miRNA which contains the miRNA that the oligomeric compound was designed to target. All compounds in Table 5 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.” The wings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR as described, supra, or they can be used in other assays to investigate the role of miRNAs or miRNA downstream targets.

TABLE 5 Chimeric phosphorothioate oligomeric  compounds having 2′-MOE wings and a deoxy  gap targeting pri-miRNAs SEQ ISIS ID Number NO Sequence pri-miRNA 328333 442 AGAACAGCATGACGTAACCT mir-140 328334 443 GCCCATCTGTGGCTTCACAG mir-30a 328335 444 GAAGTCCGAGGCAGTAGGCA mir-30a 328336 445 CTTCCTTACTATTGCTCACA mir-34 328337 446 GCTAGATACAAAGATGGAAA mir-29b-1 328338 447 CTAGACAATCACTATTTAAA mir-29b-2 328339 448 GCAGCGCAGCTGGTCTCCCC mir-29b-2 328340 449 TAATATATATTTCACTACGC mir-16-3 328341 450 TGCTGTATCCCTGTCACACT mir-16-3 328342 451 CAATTGCGCTACAGAACTGT mir-203 328343 452 TCGATTTAGTTATCTAAAAA mir-7-1 328344 453 CTGTAGAGGCATGGCCTGTG mir-7-1 328345 454 TGACTATACGGATACCACAC mir-10b 328346 455 GGAACAAGGCCAATTATTGC mir-128a 328347 456 AGAAATGTAAACCTCTCAGA mir-128a 328348 457 AGCTGTGAGGGAGAGAGAGA mir-153-1 328349 458 CTGGAGTGAGAATACTAGCT mir-153-1 328350 459 ACTGGGCTCATATTACTAGC mir-153-2 328351 460 TTGGATTAAATAACAACCTA hypothetical  miRNA-013 328352 461 CCCGGAGACAGGGCAAGACA hypothetical  miRNA-013 328353 462 AAAGCGGAAACCAATCACTG mir-27b 328354 463 GTCCCCATCTCACCTTCTCT mir-27b 328355 464 TCAGAGCGGAGAGACACAAG mir-96 328356 465 TAGATGCACATATCACTACC mir-17as/ mir-91 328357 466 CTTGGCTTCCCGAGGCAGCT mir-17as/ mir-91 328358 467 AGTTTGAAGTGTCACAGCGC mir-123/ mir-126as 328359 468 GCGTTTTCGATGCGGTGCCG mir-123/ mir-126as 328360 469 GAGACGCGGGGGCGGGGCGC mir-132 328361 470 TACCTCCAGTTCCCACAGTA mir-132 328362 471 TGTGTTTTCTGACTCAGTCA mir-108-1 328363 472 AGAGCACCTGAGAGCAGCGC mir-23b 328364 473 TCTTAAGTCACAAATCAGCA mir-23b 328365 474 TCTCCACAGCGGGCAATGTC let-7i 328366 475 GGCGCGCTGTCCGGGCGGGG mir-212 328367 476 ACTGAGGGCGGCCCGGGCAG mir-212 328368 477 GTCCTCTTGCCCAAGCAACA hypothetical  miRNA-023 328369 478 GAAGACCAATACACTCATAC mir-131-2 328370 479 CCGAGGGGCAACATCACTGC let-7b 328371 480 TCCATAGCTTAGCAGGTCCA mir-1d 328372 481 TTTGATAGTTTAGACACAAA mir-122a 328373 482 GGGAAGGATTGCCTAGCAGT mir-122a 328374 483 AGCTTTAGCTGGGTCAGGAC mir-22 328375 484 TACCATACAGAAACACAGCA mir-92-1 328376 485 TCACAATCCCCACCAAACTC mir-92-1 328377 486 TCACTCCTAAAGGTTCAAGT hypothetical  miRNA-030 328378 487 CACCCTCCAGTGCTGTTAGT mir-142 328379 488 CTGACTGAGACTGTTCACAG mir-183 328380 489 CCTTTAGGGGTTGCCACACC hypothetical  miRNA-033 328381 490 ACAGGTGAGCGGATGTTCTG mir-214 328382 491 CAGACTCCCAACTGACCAGA mir-143 328383 492 AGAGGGGAGACGAGAGCACT mir-192-1 328384 493 TCACGTGGAGAGGAGTTAAA hypothetical  miRNA-039 328385 494 AGTGCTAATACTTCTTTCAT hypothetical  miRNA-040 328386 495 ACCTGTGTAACAGCCGTGTA hypothetical  miRNA-041 328387 496 TTATCGGAACTTCACAGAGA hypothetical  miRNA-041 328388 497 TCCCATAGCAGGGCAGAGCC let-7a-3 328389 498 GGCACTTCATTGCTGCTGCC hypothetical  miRNA-043 328390 499 GGAGCCTTGCGCTCAGCATT hypothetical  miRNA-043 328391 500 ATGGTAATTTCATTTCAGGC hypothetical  miRNA-044 328392 501 GATTGCACATCCACACTGTC hypothetical  miRNA-044 328393 502 GCTGGCCTGATAGCCCTTCT mir-181a 328394 503 GTTTTTTCAAATCCCAAACT mir-181a 328395 504 CCCAGTGGTGGGTGTGACCC let-7a-1 328396 505 CTGGTTGGGTATGAGACAGA mir-205 328397 506 TTGATCCATATGCAACAAGG mir-103-1 328398 507 GCCATTGGGACCTGCACAGC mir-26a 328399 508 ATGGGTACCACCAGAACATG mir-33a 328400 509 AGTTCAAAACTCAATCCCAA mir-196-2 328401 510 GCCCTCGACGAAAACCGACT mir-196-2 328402 511 TTGAACTCCATGCCACAAGG mir-107 328403 512 AGGCCTATTCCTGTAGCAAA mir-106 328404 513 GTAGATCTCAAAAAGCTACC mir-106 328405 514 CTGAACAGGGTAAAATCACT let-7f-1 328406 515 GCAAGTCTACTCCTCAGGG let-7f-1 328407 516 AATGGAGCCAAGGTGCTGCC hypothetical  miRNA-055 328408 517 TAGACAAAAACAGACTCTGA mir-29c 328409 518 GCTAGTGACAGGTGCAGACA mir-130a 328410 519 GGGCCTATCCAAAGTGACAG hypothetical  miRNA-058 328411 520 TACCTCTGCAGTATTCTACA hypothetical  miRNA-058 328412 521 TTTACTCATACCTCGCAACC mir-218-1 328413 522 AATTGTATGACATTAAATCA mir-124a-2 328414 523 CTTCAAGTGCAGCCGTAGGC mir-124a-2 328415 524 TGCCATGAGATTCAACAGTC mir-21 328416 525 ACATTGCTATCATAAGAGCT mir-16-1 328417 526 TAATTTTAGAATCTTAACGC mir-16-1 328418 527 AGTGTCTCATCGCAAACTTA mir-144 328419 528 TGTTGCCTAACGAACACAGA mir-221 328420 529 GCTGATTACGAAAGACAGGA mir-222 328421 530 GCTTAGCTGTGTCTTACAGC mir-30d 328422 531 GAGGATGTCTGTGAATAGCC mir-30d 328423 532 CCACATATACATATATACGC mir-19b-2 328424 533 AGGAAGCACACATTATCACA mir-19b-2 328425 534 GACCTGCTACTCACTCTCGT mir-128b 328426 535 GGTTGGCCGCAGACTCGTAC hypothetical  miRNA-069 328427 536 GATGTCACTGAGGAAATCAC hypothetical  miRNA-070 328428 537 TCAGTTGGAGGCAAAAACCC hypothetical  miRNA-071 328429 538 GGTAGTGCAGCGCAGCTGGT mir-29b-3 328430 539 CCGGCTATTGAGTTATGTAC mir-129-2 328431 540 ACCTCTCAGGAAGACGGACT mir-133b 328432 541 GAGCATGCAACACTCTGTGC hypothetical  miRNA-075 328433 542 CCTCCTTGTGGGCAAAATCC let-7d 328434 543 CGCATCTTGACTGTAGCATG mir-15b 328435 544 TCTAAGGGGTCACAGAAGGT mir-29a-1 328436 545 GAAAATTATATTGACTCTGA mir-29a-1

Example 8 Chimeric Phosphorothioate Compounds Having 2′-MOE Wings and a Deoxy Gap Targeting Pri-miRNAs

In accordance with the present invention, a second series of oligomeric compounds were designed and synthesized to target different regions of pri-miRNA structures. The compounds are shown in Table 6. “Pri-miRNA” indicates the particular pri-miRNA which contains the miRNA that the oligomeric compound was designed to target. All compounds in Table 6 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.” The wings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR as described, supra, or they can be used in other assays to investigate the role of miRNAs or miRNA downstream targets.

TABLE 6 Chimeric phosphorothioate oligomeric  compounds having 2′-MOE wings  and a deoxy gap targeting pri-miRNAs SEQ ISIS ID  Number NO Sequence pri-miRNA 328637 546 GGTTCCTAATTAAACAACCC hypothetical  miRNA-079 328638 547 CCGAGGGTCTAACCCAGCCC mir-199b 328639 548 GACTACTGTTGAGAGGAACA mir-129-1 328640 549 TCTCCTTGGGTGTCCTCCTC let-7e 328641 550 TGCTGACTGCTCGCCCTTGC hypothetical  miRNA-083 328642 551 ACTCCCAGGGTGTAACTCTA let-7c 328643 552 CATGAAGAAAGACTGTAGCC mir-204 328644 553 GACAAGGTGGGAGCGAGTGG mir-145 328645 554 TGCTCAGCCAGCCCCATTCT mir-124a-1 328646 555 GCTTTTAGAACCACTGCCTC hypothetical  miRNA-088 328647 556 GGAGTAGATGATGGTTAGCC mir-213 328648 557 ACTGATTCAAGAGCTTTGTA hypothetical  miRNA-090 328649 558 GTAGATAACTAAACACTACC mir-20 328650 559 AATCCATTGAAGAGGCGATT mir-133a-1 328651 560 GGTAAGAGGATGCGCTGCTC mir-138-2 328652 561 GGCCTAATATCCCTACCCCA mir-98 328653 562 GTGTTCAGAAACCCAGGCCC mir-196-1 328654 563 TCCAGGATGCAAAAGCACGA mir-125b-1 328655 564 TACAACGGCATTGTCCTGAA mir-199a-2 328656 565 TTTCAGGCTCACCTCCCCAG hypothetical  miRNA-099 328657 566 AAAAATAATCTCTGCACAGG mir-181b 328658 567 AGAATGAGTTGACATACCAA hypothetical  miRNA-101 328659 568 GCTTCACAATTAGACCATCC mir-141 328660 569 AGACTCCACACCACTCATAC mir-131-1 328661 570 ATCCATTGGACAGTCGATTT mir-133a-2 328662 571 GGCGGGCGGCTCTGAGGCGG hypothetical  miRNA-105 328663 572 CTCTTTAGGCCAGATCCTCA hypothetical  miRNA-106 328664 573 TAATGGTATGTGTGGTGATA hypothetical  miRNA-107 328665 574 ATTACTAAGTTGTTAGCTGT mir-1b 328666 575 GATGCTAATCTACTTCACTA mir-18 328667 576 TCAGCATGGTGCCCTCGCCC mir-220 328668 577 TCCGCGGGGGCGGGGAGGCT hypothetical  miRNA-111 328669 578 AGACCACAGCCACTCTAATC mir-7-3 328670 579 TCCGTTTCCATCGTTCCACC mir-218-2 328671 580 GCCAGTGTACACAAACCAAC mir-24-2 328672 581 AAGGCTTTTTGCTCAAGGGC mir-24-1 328673 582 TTGACCTGAATGCTACAAGG mir-103-2 328674 583 TGCCCTGCTCAGAGCCCTAG mir-211 328675 584 TCAATGTGATGGCACCACCA mir-101-3 328676 585 ACCTCCCAGCCAATCCATGT mir-30b 328677 586 TCCTGGATGATATCTACCTC hypothetical  miRNA-120 328678 587 TCTCCCTTGATGTAATTCTA let-7a-4 328679 588 AGAGCGGAGTGTTTATGTCA mir-10a 328680 589 TCATTCATTTGAAGGAAATA mir-19a 328681 590 TCCAAGATGGGGTATGACCC let-7f-2 328682 591 TTTTTAAACACACATTCGCG mir-15a-1 328683 592 AGATGTGTTTCCATTCCACT mir-108-2 328684 593 CCCCCTGCCGCTGGTACTCT mir-137 328685 594 CGGCCGGAGCCATAGACTCG mir-219 328686 595 CTTTCAGAGAGCCACAGCCT mir-148b 328687 596 GCTTCCCAGCGGCCTATAGT mir-130b 328688 597 CAGCAGAATATCACACAGCT mir-19b-1 328689 598 TACAATTTGGGAGTCCTGAA mir-199b 328690 599 GCCTCCTTCATATATTCTCA mir-204 328691 600 CCCCATCTTAGCATCTAAGG mir-145 328692 601 TTGTATGGACATTTAAATCA mir-124a-1 328693 602 TTTGATTTTAATTCCAAACT mir-213 328694 603 CAAACGGTAAGATTTGCAGA hypothetical  miRNA-090 328695 604 GGATTTAAACGGTAAACATC mir-125b-1 328696 605 CTCTAGCTCCCTCACCAGTG hypothetical  miRNA-099 328697 606 GCTTGTCCACACAGTTCAAC mir-181b 328698 607 GCATTGTATGTTCATATGGG mir-1b 328699 608 TGTCGTAGTACATCAGAACA mir-7-3 328700 609 AGCCAGTGTGTAAAATGAGA mir-24-1 328701 610 TTCAGATATACAGCATCGGT mir-101-3 328702 611 TGACCACAAAATTCCTTACA mir-10a 328703 612 ACAACTACATTCTTCTTGTA mir-19a 328704 613 TGCACCTTTTCAAAATCCAC mir-15a-1 328705 614 AACGTAATCCGTATTATCCA mir-137

Example 9 Chimeric Phosphorothioate Compounds Having 2′-MOE Wings and a Deoxy Gap Targeting Pri-miRNAs

In accordance with the present invention, a third series of oligomeric compounds were designed and synthesized to target different pri-miRNA structures. The compounds are shown in Table 7. “Pri-miRNA” indicates the particular pri-miRNA which contains the miRNA that the oligomeric compound was designed to target. All compounds in Table 7 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.” The wings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR as described, supra, or they can be used in other assays to investigate the role of miRNAs or miRNA downstream targets.

TABLE 7 Chimeric phosphorothioate oligomeric  compounds having 2′-MOE wings  and a deoxy gap targeting pri-miRNAs ISIS SEQ Number ID NO Sequence pri-miRNA 328706 615 CGTGAGGGCTAGGAAATTGC mir-216 328707 616 GCAACAGGCCTCAATATCTT mir-100-1 328708 617 ACGAGGGGTCAGAGCAGCGC mir-187 328709 618 GGCAGACGAAAGGCTGACAG hypothetical  miRNA-137 328710 619 CTGCACCATGTTCGGCTCCC hypothetical  miRNA-138 328711 620 GGGGCCCTCAGGGCTGGGGC mir-124a-3 328712 621 CCGGTCCACTCTGTATCCAG mir-7-2 328713 622 GCTGGGAAAGAGAGGGCAGA hypothetical  miRNA-142 328714 623 TCAGATTGCCAACATTGTGA hypothetical  miRNA-143 328715 624 CTGGGGAGGGGGTTAGCGTC hypothetical  miRNA-144 328716 625 TGGGTCTGGGGCAGCGCAGT mir-210 328717 626 TTGAAGTAGCACAGTCATAC mir-215 328718 627 TCTACCACATGGAGTGTCCA mir-124a-3 328719 628 AGTGCCGCTGCCGCGCCGTG mir-7-2 328720 629 ACACATTGAGAGCCTCCTGA hypothetical  miRNA-142 328721 630 GTCGCTCAGTGCTCTCTAGG hypothetical  miRNA-143 328722 631 AGGCTCCTCTGATGGAAGGT hypothetical  miRNA-144 328723 632 GCTGTGACTTCTGATATTAT hypothetical  miRNA-153 328724 633 GACATCATGTGATTTGCTCA hypothetical  miRNA-154 328725 634 CACCCCAAGGCTGCAGGGCA mir-26b 328726 635 TGTCAAGCCTGGTACCACCA hypothetical  miRNA-156 328727 636 CTGCTCCAGAGCCCGAGTCG mir-152 328728 637 ACCCTCCGCTGGCTGTCCCC mir-135-1 328729 638 TAGAGTGAATTTATCTTGGT mir-135-2 328730 639 TGGTGACTGATTCTTATCCA mir-217 328731 640 CAATATGATTGGATAGAGGA hypothetical  miRNA-161 328732 641 TTTAAACACACATTCGCGCC mir-15a-2 328733 642 ACCGGGTGGTATCATAGACC let-7g 328734 643 TGCATACCTGTTCAGTTGGA hypothetical  miRNA-164 328735 644 GCCCGCCTCTCTCGGCCCCC mir-33b 328736 645 TCGCCCCCTCCCAGGCCTCT hypothetical  miRNA-166 328737 646 ACAACTGTAGAGTATGGTCA mir-16-2 328738 647 GCTGACCATCAGTACTTTCC hypothetical  miRNA-l68 328739 648 TTATAGAACAGCCTCCAGTG hypothetical  miRNA-169 328740 649 TTCAGGCACTAGCAGTGGGT hypothetical  miRNA-170 328741 650 AGTACTGCGAGGTTAACCGC hypothetical  miRNA-171 328742 651 GGACCTTTAAGATGCAAAGT hypothetical  miRNA-172 328743 652 TTCATATTATCCACCCAGGT hypothetical  miRNA-173 328744 653 CGGATCCTGTTACCTCACCA mir-182 328745 654 TGGTGCCTGCCACATCTTTG hypothetical  miRNA-175 328746 655 TGGGAGGCTGAATCAAGGAC hypothetical  miRNA-176 328747 656 TGACAACCAGGAAGCTTGTG hypothetical  miRNA-177 328748 657 GCCAGGCAGCGAGCTTTTGA hypothetical  miRNA-178 328749 658 CAGCCTGCCACCGCCGCTTT hypothetical  miRNA-179 328750 659 CTGCCCCCGTGGACCGAACA hypothetical  miRNA-180 328751 660 TCGTGCACCTGAGGAGTCTG hypothetical  miRNA-181 328752 661 CAAACGTGCTGTCTTCCTCC mir-148a 328753 662 AAGGACTCAGCAGTGTTTCA hypothetical  miRNA-183 328754 663 TCCTCGGTGGCAGAGCTCAG mir-23a 328755 664 AGACAATGAGTACACAGTTC hypothetical  miRNA-185 328756 665 CTGCAAGCACTGGTTCCCAT hypothetical  miRNA-186 328757 666 TTGCCTGAGCTGCCCAAACT mir-181c 328758 667 TCCATCACACTGTCCTATGA hypothetical  miRNA-188 328759 668 GAGGGATTGTATGAACATCT mir-216 328760 669 GCTTGTGCGGACTAATACCA mir-100-1 328761 670 GCAGGCTAAAAGAAATAAGC hypothetical  miRNA-138 328762 671 ATTGTATAGACATTAAATCA mir-124a-3 328763 672 GTTGAGCGCAGTAAGACAAC mir-7-2 328764 673 AGATGTTTCTGGCCTGCGAG hypothetical  miRNA-142 328765 674 GACAAACTCAGCTATATTGT mir-215 328766 675 ACGGCTCTGTGGCACTCATA mir-131-3 328767 676 GCTTTCTTACTTTCCACAGC mir-30c 328768 677 TACCTTTAGAATAGACAGCA mir-101-1 328769 678 AGGCTGGACAGCACACAACC mir-26b 328770 679 AGCAGGAGCCTTATCTCTCC hypothetical  miRNA-156 328771 680 ATGAGTGAGCAGTAGAATCA mir-135-1 328772 681 TGAGACTTTATTACTATCAC mir-135-2 328773 682 TACTTTACTCCAAGGTTTTA mir-15a-2 328774 683 GCACCCGCCTCACACACGTG mir-33b 328775 684 TTCCCGACCTGCCTTTACCT hypothetical  miRNA-166 328776 685 TCCTGTAATTATAGGCTAGC hypothetical  miRNA-169 328777 686 GGATCATATCAATAATACCA hypothetical  miRNA-172 328778 687 TGCTGAGACACACAATATGT hypothetical  miRNA-176 328779 688 TGTTTGTCTCCAAGAAACGT hypothetical  miRNA-177 328780 689 TGTCATGGACAGGATGAATA hypothetical  miRNA-179 328781 690 TCTATCATACTCAGAGTCGG mir-148a 328782 691 TTGTGACAGGAAGCAAATCC mir-23a 328783 692 CATCAGAGTCACCAACCCCA hypothetical  miRNA-185 328784 693 CAAGAGATGTCTCGTTTTGC hypothetical  miRNA-186

Example 10 Chimeric Phosphorothioate Compounds Having 2′-MOE Wings and a Deoxy Gap Targeted to the Stem Loop of Pri-miRNA Structures

In accordance with the present invention, a fourth series of oligomeric compounds were designed to target the stem loop of different pri-miRNA structures. In some cases, these oligomeric compounds contain mismatches, and thus hybridize with partial complementarity to the stem loop structure of the pri-miRNA targeted. The compounds are shown in Table 8. “Pri-miRNA” indicates the particular pri-miRNA that the oligomeric compound was designed to target. All compounds in Table 8 are chimeric oligonucleotides (“gapmers”), composed of a central “gap” region consisting of 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by “wings.” The wings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR as described, supra, or they can be used in other assays to investigate the role of miRNAs or downstream nucleic acid targets.

TABLE 8 Chimeric phosphorothioate compounds having 2′-MOE wings and a deoxy gap targeted to the stem loop of pri-miRNA structures Compound SEQ ID Number NO. Sequence Pri-miRNA RG1 694 GTGGTAGAACAGCATGACGTC mir-140 RG2 695 AGCTGTGAAGCCACGATGGGC mir-30a RG3 696 AGATACAAAGATGGAAAAATC mir-29b-1 RG4 697 CTTCCTTACTATTGCTCACAA mir-34 RG5 698 TGTTTAATATATATTTCACTC mir-16-3 RG6 699 TGTCAAGACATCGCGTTAACA mir-203 RG7 700 TGTCGATTTAGTTATCCAACA mir-7-1 RG8 701 GTGACTATACGGATACCACAC mir-10b RG9 702 ACCTCTCCAAATGTAAAGA mir-128a RG10 703 CAAAGCGGAAACCAATCACTG mir-27b RG11 704 CTGCAGTACATGCACATATCA mir-91 RG12 705 AACAATGACACCCTTGACCT mir-132 RG13 706 TTTTAATCTTAAGTCACAAA mir-23b RG14 707 ATCTCCACAGCGGGCAATGTC let-7i RG15 708 TATGAAGACCAATACACTCCA mir-131-2 RG16 709 GGGGCAACATCACTGCCC let-7b RG17 710 CCATGTTAGCAGGTCCATATG mir-1d RG18 711 GTTTGATAGTTTAGACACAAA mir-122a RG19 712 TGGGTCAGGACTAAAGCTTC mir-22 RG20 713 AATACCATACAGAAACACAGC mir-92-1 RG21 714 TTCGTGATGATTGTCGTGCC mir-142 RG22 715 ACTGCGAGACTGTTCACAGTT mir-183 RG23 716 TACAGGTGAGCGGATGTTCTG mir-214 RG24 717 TCTCAGCTCCCAACTGACCAG mir-143 RG25 718 ACCGCAGATATTACAGCCACT let-7a-3 RG26 719 CCTGATAGCCCTTCTTAAGGA mir-181a RG27 720 CTTGATCCATATGCAACAAGG mir-103-1 RG28 721 GCCATTGGGACCTGCACACC mir-26a RG29 722 GCATGGGTACCACCCCATGC mir-33a RG30 723 CGAGTTCAAAACTCAATCCCA mir-196-2 RG31 724 CTTGAACTCCATGCCACAAGG mir-107 RG32 725 GTAGATCTCAAAAAGCTAGC mir-106 RG33 726 GAACAGGGTAAAATCACTAC let-7f-1 RG34 727 AGACAAAAACAGACTCTGAA mir-29c RG35 728 GCTAGTGACAGGTCCAGACAG mir-130a RG36 729 TTTACTCATACCTCGCAACCA mir-218-1 RG37 730 TTAATTGTATGACATTAAATCA mir-124a-2 RG38 731 TGCCATGAGATTCAACAGTCA mir-21 RG39 732 GATAATATTTAGAATCTTAAC mir-16-1 RG40 733 TAGTGTCTCATCGCAAACTTA mir-144 RG41 734 CTGTTGCCTAACGAACACAGA mir-221 RG42 735 TGCTGATTACGAAAGACAGGAT mir-222 RG43 736 GCTTAGCTGTGTCTTACAGCT mir-30d

Example 11 Effects of Oligomeric Compounds Targeting miRNAs on Apoptosis in Caspase Assay

Programmed cell death or apoptosis involves the activation of proteases, a family of intracellular proteases, through a cascade which leads to the cleavage of a select set of proteins. The caspase family contains at least 14 caspases, with differing substrate preferences. The caspase activity assay uses a DEVD peptide to detect activated caspases in cell culture samples. The peptide is labeled with a fluorescent molecule, 7-amino-4-trifluoromethyl coumarin (AFC). Activated caspases cleave the DEVD peptide resulting in a fluorescence shift of the AFC. Increased fluorescence is indicative of increased caspase activity and consequently increased cell death. The chemotherapeutic drugs taxol, cisplatin, etoposide, gemcitabine, camptothecin, aphidicolin and 5-fluorouracil all have been shown to induce apoptosis in a caspase-dependent manner.

The effect of several oligomeric compounds of the present invention was examined in cells expressing miRNA targets. The cells expressing the targets used in these experiments were T47D, a breast carcinoma cell line. Other cell lines can also be employed in this assay and these include normal human mammary epithelial cells (HMECs) as well as two breast carcinoma cell lines, MCF7 and T47D. All of the cell lines were obtained from the American Type Culture Collection (Manassas, Va.). The latter two cell lines express similar genes but MCF7 cells express the tumor suppressor p53, while T47D cells are deficient in p53. MCF-7 cells are routinely cultured in DMEM low glucose (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. T47D cells were cultured in Gibco DMEM High glucose media supplemented with 10% Fetal Bovine Serum (FBS).

Cells were plated at 10,000 cells per well for HMEC cells or 20,000 cells per well for MCF7 and T47D cells, and allowed to attach to wells overnight. Plates used were 96 well Costar plate 1603 (black sides, transparent bottom). DMEM high glucose medium, with and without phenol red, were obtained from Invitrogen (San Diego, Calif.). MEGM medium, with and without phenol red, were obtained from Biowhittaker (Walkersville, Md.). The caspase-3 activity assay kit was obtained from Calbiochem (Cat. #HTS02) (EMD Biosciences, San Diego, Calif.).

Before adding to cells, the oligomeric compound cocktail was mixed thoroughly and incubated for 0.5 hrs. The oligomeric compound or the LIPOFECTIN™-only vehicle control was added (generally from a 3 μM stock of oligonucleotide) to a final concentration of 200 nM with 6 μg/ml LIPOFECTIN™. The medium was removed from the plates and the plates were tapped on sterile gauze. Each well was washed in 150 μl of PBS (150 μL HBSS for HMEC cells). The wash buffer in each well was replaced with 100 μL of the oligomeric compound/OPTI-MEM™/LIPOFECTIN™ cocktail (this was T=0 for oligomeric compound treatment). The plates were incubated for 4 hours at 37° C., after which the medium was dumped and the plate was tapped on sterile gauze. 100 μl of full growth medium without phenol red was added to each well. After 48 hours, 50 μl of oncogene buffer (provided with Calbiochem kit) with 10 μM DTT was added to each well. 20 μl of oncogene substrate (DEVD-AFC) was added to each well. The plates were read at 400±25 nm excitation and 508±20 nm emission at t=0 and t=3 time points. The t=0×(0.8) time point was subtracted from the t=3 time point, and the data are shown as percent of LIPOFECTIN™-only (untreated control) treated cells.

Four experiments were performed and the results are shown in Tables 9-12. The concentration of oligomeric compound used was 200 nM. All compounds in Tables 9-12 are chimeric oligomeric compounds (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.” The wings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the compound. All cytidine residues are 5-methylcytidines. As a control a 20-mer oligonucleotide random-mer, ISIS-29848 (NNNNNNNNNNNNNNNNNNNN; where N is A, T, C or G; herein incorporated as SEQ ID NO: 737) was used. In addition, two positive controls targeting expressed genes known to induce apoptosis when inhibited were included. These were ISIS-148715 (TTGTCCCAGTCCCAGGCCTC; herein incorporated as SEQ ID NO: 738) which targets human Jagged2 and ISIS-226844 (GCCCTCCATGCTGGCACAGG; herein incorporated as SEQ ID NO: 739) which targets human Notch1. Both positive controls have the same chemistry and gap structure as the compounds being tested. An increase in fluorescence indicates that the compound, by inhibiting its target, induces apoptosis as compared to untreated controls (UTC).

TABLE 9 Effects of oligomeric compounds targeting miRNAs on Apoptosis in Caspase Assay SEQ ID Fold Increase ISIS Number NO. Pri-miRNA over UTC UTC N/A N/A 1.0 Untreated control ISIS-29848 737 N/A 3.5 n-mer ISIS-148715 738 Jagged2 1.5 Positive control ISIS-226844 739 Notch1 3.6 Positive control 328371 480 mir-1d 1.2 328400 509 mir-196-2 1.3 328420 529 mir-222 1.0 328692 601 mir-124a-1 1.2 328381 490 mir-214 1.1 328691 600 mir-145 0.9 328391 500 hypothetical miRNA-044 0.8 328415 524 mir-21 1.1 328433 542 let-7d 1.0 328643 552 mir-204 0.9 328377 486 hypothetical miRNA-030 0.7 328405 514 let-7f-1 1.0 328372 481 mir-122a 1.0 328403 512 mir-106 1.0 328424 533 mir-19b2 0.9 328648 557 hypothetical miRNA-090 1.1 328397 506 mir-103-1 1.2 328656 565 hypothetical miRNA-099 1.1 328392 501 hypothetical miRNA-044 1.0 328421 530 mir-30d 1.2 328417 526 mir-16-1 1.0 328647 556 mir-213 0.9 328378 487 mir-142 1.0 328416 525 mir-16-1 0.9

TABLE 10 Effects of oligomeric compounds targeting miRNAs on Apoptosis in Caspase Assay SEQ ID Fold Increase ISIS Number NO. Pri-miRNA over UTC UTC N/A N/A 0.9 Untreated control ISIS-29848 737 N/A 3.0 n-mer ISIS-148715 738 Jagged2 1.0 Positive control ISIS-226844 739 Notch1 3.1 Positive control 328375 484 mir-92-1 0.9 328382 491 mir-143 0.9 328383 492 mir-192-1 1.2 328385 494 hypothetical miRNA-040 0.9 328395 504 let-7a-1 1.0 328398 507 mir-26a 0.9 328399 508 mir-33a 1.0 328402 511 mir-107 1.2 328408 517 mir-29c 0.9 328409 518 mir-130a 0.7 328422 531 mir-30d 1.0 328423 532 mir-19b-2 0.6 328425 534 mir-128b 0.8 328431 540 mir-133b 0.9 328436 545 mir-29a-1 0.9 328646 555 hypothetical miRNA-088 1.1 328649 558 mir-20 1.0 328651 560 mir-138-2 0.9 328652 561 mir-98 1.2 328657 566 mir-181b 0.8 328672 581 mir-24-1 0.9 328694 603 hypothetical miRNA-090 0.8 328696 605 hypothetical miRNA-099 1.5 328700 609 mir-24-1 0.8

TABLE 11 Effects of oligomeric compounds targeting miRNAs on Apoptosis in Caspase Assay SEQ ID Fold Increase ISIS Number NO. Pri-miRNA over UTC UTC N/A N/A 0.9 Untreated control ISIS-29848 737 N/A 3.2 n-mer ISIS-148715 738 Jagged2 1.1 Positive control ISIS-226844 739 Notch1 3.1 Positive control 328374 483 mir-22 1.1 328376 485 mir-92-1 0.7 328384 493 hypothetical miRNA-039 1.0 328386 495 hypothetical miRNA-041 0.7 328390 499 hypothetical miRNA-043 0.9 328393 502 mir-181a 1.5 328404 513 mir-106 0.9 328406 515 let-7f-1 1.0 328407 516 hypothetical miRNA-055 1.2 328410 519 hypothetical miRNA-058 1.5 328411 520 hypothetical miRNA-058 0.8 328413 522 mir-124a-2 0.8 328426 535 hypothetical miRNA-069 1.3 328427 536 hypothetical miRNA-070 0.8 328435 544 mir-29a-1 1.3 328637 546 hypothetical miRNA-079 1.0 328638 547 mir-199b 0.8 328639 548 mir-129-1 0.8 328645 554 mir-124a-1 2.2 328653 562 mir-196-1 1.1 328654 563 mir-125b-1 1.0 328655 564 mir-199a-2 0.7 328689 598 mir-199b 0.8 328695 604 mir-125b-1 0.8

TABLE 12 Effects of oligomeric compounds targeting miRNAs on Apoptosis in Caspase Assay SEQ ID Fold Increase ISIS Number NO. Pri-miRNA over UTC UTC N/A N/A 1.0 Untreated control ISIS-29848 737 N/A 3.5 n-mer ISIS-148715 738 Jagged2 1.3 Positive control ISIS-226844 739 Notch1 3.5 Positive control 328373 482 mir-122a 0.9 328379 488 mir-183 1.1 328387 496 hypothetical miRNA-041 1.4 328388 497 let-7a-3 0.9 328389 498 hypothetical miRNA-043 1.1 328394 503 mir-181a 0.8 328396 505 mir-205 0.8 328401 510 mir-196-2 0.8 328412 521 mir-218-1 1.2 328414 523 mir-124a-2 0.9 328418 527 mir-144 1.0 328419 528 mir-221 0.7 328430 539 mir-129-2 1.3 328432 541 hypothetical miRNA-075 0.6 328434 543 mir-15b 0.8 328640 549 let-7e 0.9 328641 550 hypothetical miRNA-083 1.1 328642 551 let-7c 1.0 328644 553 mir-145 0.7 328650 559 mir-133a-1 0.8 328658 567 hypothetical miRNA-101 1.2 328690 599 mir-204 0.8 328693 602 mir-213 1.0 328697 606 mir-181b 1.0

From these data, it is evident that SEQ ID NOs. 480, 509, 601, 490, 524, 557, 506, 565, 530, 605, 492, 561, 511, 555, 483, 502, 535, 562, 544, 519, 516, 554, 496, 567, 521, 539, 488, 498, and 550 induce apoptosis in T47D cells, while SEQ ID NOs. 500, 486, 518, 532, 534, 566, 603, 609, 485, 495, 520, 522, 536, 547, 548, 564, 598, 604, 503, 505, 510, 528, 541, 543, 553, 559, and 599 prevent or have a protective effect from apoptosis in the same system.

Example 12 Oligomeric Compounds Targeting the Mir-30a Pri-miRNA Structure

In one embodiment of the invention, oligomeric compounds targeting the hairpin structure of mir-30a pri-miRNA were designed and tested for their effects on miRNA signaling in 293T cells (American Type Culture Collection (Manassas, Va.)).

A synthetic DNA fragment comprised of four tandem repeats of the target site for mir-30a was cloned into the vector pGL3-C (purchased from Promega Corp., Madison Wis.) at the unique XbaI site (pGL3C-M30-4X). This places the target site in the 3′UTR of the luciferase reporter vector. An oligomeric compound mimicking the mir-30a pri-miRNA (AATTTAATACGACTCACTATAGGGCGACTGTAAACATCCTCGACTGGAAGCTGTGAAG CCACAGATGGGCTTTCAGTCGGATGTTTGCAGCTGC, herein incorporated as SEQ ID NO: 1749) was in vitro transcribed using T7 RNA polymerase and a DNA template produced by PCR (the T7 promoter is shown in bold).

On the day prior to the experiment 24-well plates were seeded with 293T cells at 50% confluency. The following morning cells were treated with oligomeric compounds targeted to the mir-30a pri-miRNA mimic. The oligomeric compounds used in this study are shown in Table 13. All of the compounds are 20 nucleobases in length having either a phosphorothioate backbone throughout (PS) or a phosphodiester backbone throughout (PO). As designated in the table, ISIS 328076, 328078, 328081, 328084, 328086, 328088 are chimeric oligomeric compounds (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.” The wings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides. All cytidine residues are 5-methylcytidines. The remaining compounds in the table have 2′-methoxyethoxy (MOE) nucleotides throughout with either a phosphorothioate (PS) or phosphodiester (PO) internucleoside linkages.

If the compound targeted the pre-loop of the mir-30a pri-miRNA structure, that designation is also noted in the table.

TABLE 13 Oligomeric compounds targeting the mir-30a pri-miRNA SEQ Isis ID Number Sequence Chemistry NO 328075 GCTTCACAGCTTCCAGTCGA (PS/MOE) 740 328076 GCTTCACAGCTTCCAGTCGA (PS/MOE 5-10-5 740 gapmer) 328077 CCCATCTGTGGCTTCACAGC (PS/MOE); pre-loop 741 328078 CCCATCTGTGGCTTCACAGC (PS/MOE 5-10-5 741 gapmer); pre-loop 328079 CCCATCTGTGGCTTCACAGC (PO/MOE); pre-loop 741 328080 TGAAAGCCCATCTGTGGCTT (PS/MOE); pre-loop 742 328081 TGAAAGCCCATCTGTGGCTT (PS/MOE 5-10-5 742 gapmer); pre-loop 328082 TGAAAGCCCATCTGTGGCTT (PO/MOE); pre-loop 742 328083 GCAGCTGCAAACATCCGACT (PS/MOE) 743 328084 GCAGCTGCAAACATCCGACT (PS/MOE 5-10-5 743 gapmer) 328085 CATCTGTGGCTTCACAGCTT (PS/MOE) 744 328086 CATCTGTGGCTTCACAGCTT (PS/MOE 5-10-5 744 gapmer) 328087 AAGCCCATCTGTGGCTTCAC (PS/MOE) 745 328088 AAGCCCATCTGTGGCTTCAC (PS/MOE 5-10-5 745 gapmer)

Cells were washed once with PBS then oligomeric compounds were added to triplicate wells at 150 nM in OPTI-MEM™ media and 4.5 μl/ml LIPOFECTIN™ reagent (Invitrogen Corporation, Carlsbad, Calif.). After 3 hours, the media was removed, and the cells were treated with the mir-30a pri-miRNA mimic at 200 nM in OPTI-MEM™ with 6 μl/ml LIPOFECTIN™ reagent. After 3 hours the media was removed from the cells. The reporter plasmid, pGL3C-M30-4X, was then transfected using SuperFect reagent. 20 μg of pGL3C-M30-4X and 2 μg of pRL-CMV, a plasmid expressing Renilla luciferase, were suspended in 600 μL of serum-free DMEM to which 120 μl of Superfect was added. After a 5 minute incubation, 6 mls of DMEM+10% FCS was added. 125 μL of the plasmid/SuperFect suspension was added to each well. After a 2 hour incubation cells were washed and fresh growth media added. Cells were incubated overnight.

The following morning the media was removed and the cells were lysed in 120 μL passive lysis buffer (PLB; Promega). 40 μL of the lysate was then assayed for Photinus (PL) and Renilla (RL) luciferases using a Dual Luciferase Assay kit (Promega) according to the manufacturer's protocol. The results below are given as percent pGL3C-M30-4× expression (PL) normalized to pRL-CMV expression (RL). The 20-nucleobase oligonucleotide random-mer ISIS Number 29848 was used as a negative control. The data are shown in Table 14.

TABLE 14 Effects of oligomeric compounds targeting the mir-30a pri-miRNA on reporter gene expression percent control SEQ ID luciferase NO ISIS Number expression N/A Untreated control 100 N/A Mir-30a pri-miRNA only 62 737 29848 control added 63 after mir-30a pri-miRNA 292 327874 66 740 328075 55 740 328076 57 741 328077 70 741 328078 63 742 328080 72 742 328081 80 743 328084 75 744 328085 72 744 328086 95 745 328087 83 745 328088 107

Upon administration of the mir-30a pri-miRNA mimic, the pri-miRNA is believed to be processed in the cell by the endogenous Drosha RNase III enzyme into a pre-miRNA, which is then processed by human Dicer into a mature miRNA, which is then able to hybridize to the target site, thus effectively reducing luciferase reporter expression.

Upon treatment of the system with the oligomeric compounds targeting the mir-30a pri-miRNA, the processing and/or production of the mir-30a mature miRNA is inhibited, and the mir-30a miRNA is no longer able to bind its target site, thus allowing luciferase reporter expression to increase.

Cells treated with mir-30a pri-miRNA mimic show an approximately 38% reduction in luciferase expression compared to the untreated controls. Treatment with ISIS 328086, 328087 and 328088 had the most dramatic effect in reversing the mir-30a miRNA-mediated silencing, restoring luciferase reporter expression to near control levels. Thus, it was demonstrated that the oligomeric compound mimicking the mir-30a pri-miRNA silences luciferase activity from the reporter vector, and that oligomeric compounds targeting the mir-30a pri-miRNA can inhibit its silencing activity, possibly by interfering with its processing into the pre-miRNA or mature miRNA molecules.

ISIS 328085 to ISIS 328088 were designed to target the mir-30a pri-miRNA as pseudo half-knot compounds. Methods for the preparation of pseudo half-knot compounds are disclosed in U.S. Pat. No. 5,512,438 which is incorporated herein by reference. This motif has been used to disrupt the structure of regulatory RNA stem loops in larger viral genomic structures. (Ecker et al, Science. 1992; 257:958-61). However, this is the first example of the pseudo half-knot motif being used to regulate a small non-coding RNA, more specifically a miRNA such as those disclosed herein. It is also the first demonstration of apoptotic modulation in a cell by pseudo half-knot structured oligomeric compounds.

Example 13 Effects of Oligomeric Compounds Targeting miRNAs on Expression of Adipocyte Differentiation Markers

The effect of several oligomeric compounds of the present invention targeting miRNAs on the expression of markers of cellular differentiation was examined in preadipocytes.

One of the hallmarks of cellular differentiation is the upregulation of gene expression. During adipocyte differentiation, the gene expression patterns in adipocytes change considerably. An excessive recruitment and differentiation of preadipocytes into mature adipocytes is a characteristic of human obesity, which is a strong risk factor for type 2 diabetes, hypertension, atherosclerosis, cardiovascular disease, and certain cancers. Some genes known to be upregulated during adipocyte differentiation include hormone-sensitive lipase (HSL), adipocyte lipid binding protein (aP2), glucose transporter 4 (Glut4), and PPARγ (Peroxisome proliferator-activated receptor gamma). These genes play important roles in the uptake of glucose and the metabolism and utilization of fats. For example, HSL is involved in the mobilization of fatty acids from adipose tissue into the bloodstream; studies suggest that increased free fatty acid levels are one of the causative factors in type 2 diabetes. aP2 is believed to play a role in atherosclerosis. Glut4 is important in insulin signaling. PPARγ is believed to be involved in adipocyte differentiation, insulin sensitivity, and colonic tumor development.

Leptin is also a marker for differentiated adipocytes. In the adipocyte assay, leptin secretion into the media above the differentiated adipocytes was measured by protein ELISA. Cell growth, transfection and differentiation procedures were carried out as described for the Triglyceride accumulation assay (see below). On day nine post-transfection, 96-well plates were coated with a monoclonal antibody to human leptin (R&D Systems, Minneapolis, Minn.) and left at 4° C. overnight. The plates were blocked with bovine serum albumin (BSA), and a dilution of the media was incubated in the plate at RT for 2 hours. After washing to remove unbound components, a second monoclonal antibody to human leptin (conjugated with biotin) was added. The plate was then incubated with strepavidin-conjugated horseradish peroxidase (HRP) and enzyme levels are determined by incubation with 3,3′,5,5′-Tetramethlybenzidine, which turns blue when cleaved by HRP. The OD₄₅₀ was read for each well, where the dye absorbance is proportional to the leptin concentration in the cell lysate. Results are expressed as a percent±standard deviation relative to transfectant-only controls.

An increase in triglyceride content is another well-established marker for adipocyte differentiation. The triglyceride accumulation assay measures the synthesis of triglyceride by adipocytes. Triglyceride accumulation was measured using the Infinity™ Triglyceride reagent kit (Sigma-Aldrich, St. Louis, Mo.). Human white preadipocytes (Zen-Bio Inc., Research Triangle Park, N.C.) were grown in preadipocyte media (ZenBio Inc.). One day before transfection, 96-well plates were seeded with 3000 cells/well. Cells were transfected according to standard published procedures with 250 nM oligomeric compound in LIPOFECTIN™ (Invitrogen Corporation, Carlsbad, Calif.) (Monia et al., J. Biol. Chem. 1993 268(19):14514-22). Oligomeric compounds were tested in triplicate on each 96-well plate, and the effects of TNF-α, a positive drug control that inhibits adipocyte differentiation, were also measured in triplicate. Negative and transfectant-only controls may be measured up to six times per plate. After the cells have reached confluence (approximately three days), they were exposed to differentiation media (Zen-Bio, Inc.) containing a PPAR-γ agonist, IBMX, dexamethasone, and insulin for three days. Cells were then fed adipocyte media (Zen-Bio, Inc.), which was replaced at 2 to 3 day intervals. On day nine post-transfection, cells were washed and lysed at room temperature, and the triglyceride assay reagent was added. Triglyceride accumulation was measured based on the amount of glycerol liberated from triglycerides by the enzyme lipoprotein lipase. Liberated glycerol is phosphorylated by glycerol kinase, and hydrogen peroxide is generated during the oxidation of glycerol-1-phosphate to dihydroxyacetone phosphate by glycerol phosphate oxidase. Horseradish peroxidase (HRP) uses H₂O₂ to oxidize 4-aminoantipyrine and 3,5 dichloro-2-hydroxybenzene sulfonate to produce a red-colored dye. Dye absorbance, which is proportional to the concentration of glycerol, was measured at 515 nm using an UV spectrophotometer. Glycerol concentration was calculated from a standard curve for each assay, and data were normalized to total cellular protein as determined by a Bradford assay (Bio-Rad Laboratories, Hercules, Calif.). Results are expressed as a percent±standard deviation relative to transfectant-only control.

For assaying adipocyte differentiation, expression of the four hallmark genes, HSL, aP2, Glut4, and PPARγ, as well as triglyceride (TG) accumulation and leptin secretion were measured in adipocytes transfected with the uniform 2′-MOE phosphorothioate (PS) oligomeric compounds previously described. Cells are lysed on day nine post-transfection, in a guanadinium-containing buffer and total RNA is harvested. Real-time PCR is performed (Applied Biosystems, Prism 7700) on the total RNA using the following primer/probe sets for the adipocyte differentiation hallmark genes: (aP2): forward 5′-GGTGGTGGAATGCGTCATG-3′ (SEQ ID NO: 746), reverse 5′-CAACGTCCCTTGGCTTATGC-3′ (SEQ ID NO: 747), probe 5′-FAM-AAGGCGTCACTTCCACGAGAGTTTATGAGA-TAMRA-3′ (SEQ ID NO: 748); (Glut4): forward 5′-GGCCTCCGCAGGTTCTG-3′ (SEQ ID NO: 749), reverse 5′-TTCGGAGCCTATCTGTTGGAA-3′ (SEQ ID NO: 750), probe 5′-FAM-TCCAGGCCGGAGTCAGAGACTCCA-TAMRA-3′ (SEQ ID NO: 751); (HSL): forward 5′-ACCTGCGCACAATGACACA-3′ (SEQ ID NO: 752), reverse 5′-TGGCTCGAGAAGAAGGCTATG-3′ (SEQ ID NO: 753), probe 5′-FAM-CCTCCGCCAGAGTCACCAGCG-TAMRA-3′ (SEQ ID NO: 754); (PPAR-γ): forward 5′-AAATATCAGTGTGAATTACAGCAAACC-3′ (SEQ ID NO: 755), reverse 5′-GGAATCGCTTTCTGGGTCAA-3′ (SEQ ID NO: 756), probe 5′-FAM-TGCTGTTATGGGTGAAACTCTGGGAGATTCT-TAMRA-3′ (SEQ ID NO: 757). The amount of total RNA in each sample is determined using a Ribogreen Assay (Molecular Probes, Eugene, Oreg.), and expression levels of the adipocyte differentiation hallmark genes were normalized to total RNA. Leptin protein and triglyceride levels as well as mRNA levels for each of the four adipocyte differentiation hallmark genes are expressed relative to control levels (control=treatment with ISIS-29848 (SEQ ID NO: 737)). Results of two experiments are shown in Tables 15 and 16.

TABLE 15 Effects of oligomeric compounds targeting miRNAs on expression of adipocyte differentiation markers ISIS SEQ ID Number NO TG AP2 HSL Glut4 PPAR gamma 327876 294 0.47 0.75 0.47 0.36 0.57 327878 296 0.65 0.85 0.93 0.69 0.97 327880 298 0.52 0.97 0.80 1.11 0.53 327888 306 0.98 1.18 1.38 1.37 1.36 327889 307 0.47 0.69 0.59 0.55 0.71 327890 308 0.92 0.91 0.86 1.10 1.18 327892 310 0.42 0.31 0.25 0.07 0.32 327901 319 0.54 0.42 0.33 0.19 0.30 327903 321 1.20 1.15 1.23 1.72 1.19 327905 323 0.69 1.14 1.11 0.84 0.54 327913 331 0.59 0.99 0.92 0.84 0.72 327919 337 0.58 0.79 0.57 0.32 0.52 327922 340 1.09 0.99 0.95 1.75 1.37 327925 343 0.72 0.77 0.78 1.99 0.60 327933 351 1.48 1.46 1.35 2.52 1.52 327934 352 0.99 1.20 1.02 1.22 0.97 327939 357 0.92 1.08 1.21 0.87 0.83 327941 359 1.31 1.78 1.73 2.07 0.80 327954 372 0.58 0.95 1.03 0.92 0.73

TABLE 16 Effects of oligomeric compounds targeting miRNAs on expression of adipocyte differentiation markers SEQ ISIS ID Number NO TG Leptin AP2 HSL Glut4 PPAR gamma 327888 306 0.44 1.38 0.47 0.50 0.17 0.66 327889 307 0.46 1.05 0.57 0.54 0.46 0.82 327890 308 0.61 1.36 0.69 0.67 0.67 0.94 327893 311 0.95 1.14 0.97 0.85 1.47 1.03 327901 319 0.53 1.02 0.47 0.47 0.29 0.72 327903 321 0.58 1.61 0.92 0.80 1.12 0.98 327905 323 0.58 1.62 0.68 0.69 0.40 0.83 327919 337 0.40 1.44 0.48 0.37 0.18 0.57 327922 340 0.43 1.25 0.75 0.72 0.43 0.80 327925 343 0.63 1.40 0.77 0.75 0.61 0.83 327926 344 1.06 1.47 0.85 0.82 1.10 0.93 327930 348 0.97 0.95 0.86 0.89 1.01 0.98 327931 349 1.11 1.12 1.00 0.99 1.37 1.56 327934 352 0.62 1.25 0.66 0.64 0.44 0.72 327938 356 1.05 1.35 0.86 0.85 0.80 0.90 327939 357 0.59 2.67 0.69 0.63 0.30 0.70 327941 359 0.42 0.54 0.88 0.81 0.44 0.86 327942 360 0.85 2.03 0.82 0.79 0.66 0.87 327955 373 0.81 1.22 0.74 0.82 0.45 0.92 327967 385 0.90 1.22 0.86 0.97 0.56 0.89

From these data, values above 1.0 for triglyceride accumulation (column “TG” in the tables) indicate that the compound has the ability to stimulate triglyceride accumulation, whereas values at or below 1.0 indicate that the compound inhibits triglyceride accumulation. With respect to leptin secretion (column “Leptin” in the tables), values above 1.0 indicate that the compound has the ability to stimulate secretion of the leptin hormone, and values at or below 1.0 indicate that the compound has the ability to inhibit secretion of leptin. With respect to the four adipocyte differentiation hallmark genes (columns “AP2,” “HSL,” “Glut4,” and “PPAR gamma” in the tables), values above 1.0 indicate induction of cell differentiation, whereas values at or below 1.0 indicate that the compound inhibits differentiation.

Several compounds were found to have remarkable effects. For example, the oligomeric compounds ISIS Number 327889 (SEQ ID NO: 307), targeted to mir-23b; ISIS Number 327892 (SEQ ID NO: 310), targeted to mir-131-1, mir-131-2 and mir-131-3 (also known as mir-9); ISIS Number 327942 (SEQ ID NO: 360) targeted to mir-141 and ISIS Number 327901 (SEQ ID NO: 319), targeted to mir-143 were shown to significantly reduce the expression levels of 5 of 6 markers of adipocyte differentiation (excepting leptin levels), indicating that these oligomeric compounds have the ability to block adipocyte differentiation. Therefore, these oligomeric compounds may be useful as pharmaceutical agents with applications in the treatment, attenuation or prevention of obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases as well as having potential applications in the maintenance of the pluripotent phenotype of stem or precursor cells.

The compound ISIS Number 327939 (SEQ ID NO: 357), targeted to mir-125b-1, for example, produced surprising results in that it demonstrates a significant increase in leptin secretion but a concomitant decrease in triglyceride accumulation and a decrease in the expression of all four adipocyte differentiation hallmark genes, indicating that this oligomeric compound may be useful as a pharmaceutic agent in the treatment of obesity, as well as having applications in other metabolic diseases.

The oligomeric compound ISIS Number 327931 (SEQ ID NO: 349), targeted to let-7c is an example of a compound which demonstrates an increase in four out of six markers of adipocyte differentiation, including a significant increase in the expression of PPAR-γ. This oligomeric compound may be useful as a pharmaceutical agent in the treatment of diseases in which the induction of cell differentiation is desirable.

The oligomeric compound ISIS Number 327933 (SEQ ID NO: 351), targeted to mir-145 is an example of a compound which demonstrates an increase in all six markers of adipocyte differentiation. This oligomeric compound may be useful as a pharmaceutical agent in the treatment of diseases in which the induction of adipocyte differentiation is desirable, such as anorexia, or for conditions or injuries in which the induction of cellular differentiation is desirable, such as Alzheimers disease or central nervous system injury, in which regeneration of neural tissue (such as from pluripotent stem cells) would be beneficial. Furthermore, this oligomeric compound may be useful in the treatment, attenuation or prevention of diseases in which it is desirable to induce cellular differentiation and/or quiescence, for example in the treatment of hyperproliferative disorders such as cancer.

In some embodiments, differentiating adipocytes were treated with uniform 2′-MOE phosphorothioate oligomeric compounds according to the methods described above, and the expression of the four hallmark genes, HSL, aP2, Glut4, and PPARγ, as well as triglyceride (TG) accumulation were measured. TG levels as well as mRNA levels for each of the four adipocyte differentiation hallmark genes are expressed as a percentage of control levels (control=treatment with ISIS 342673; AGACTAGCGGTATCTTTATCCC; herein incorporated as SEQ ID NO: 758), a uniform 2′-MOE phosphorothioate oligomeric compound containing 15 mismatches with respect to the mature mir-143 miRNA). Undifferentiated adipocytes were also compared as a negative control. As a positive control, differentiating adipocytes were treated with ISIS 105990 (AGCAAAAGATCAATCCGTTA; herein incorporated as SEQ ID NO: 759), a 5-10-5 gapmer oligomeric compound targeting the PPAR-gamma mRNA, previously demonstrated to inhibit adipocyte differentiation. The effects of TNF-α, also known to inhibit adipocyte differentiation, were also measured. Results of these experiments are shown in Tables 17 and 18.

TABLE 17 Effects of oligomeric compounds targeting miRNAs on expression of adipocyte differentiation markers ISIS SEQ Number ID NO TG AP2 HSL Glut4 PPAR gamma Untreated N/A 88.5 87.8 88.6 102.7 94.9 control 105990 759 28.2 51.6 49.2 59.5 51.8 342673 758 100.0 100.0 100.0 100.0 100.0 TNF-alpha N/A 10.0 5.5 0.7 0.5 18.8 Undiff. N/A 2.7 0.0 0.3 0.1 9.2 adipocytes 328116 418 82.1 87.7 75.8 75.2 78.4 328117 419 55.0 65.4 61.7 68.1 64.1 328118 420 69.3 92.7 85.3 76.6 80.2 328119 421 90.2 99.9 98.5 95.2 82.7 328120 422 82.7 81.0 77.7 94.8 70.5 328121 423 134.8 127.0 126.0 140.8 103.6 328122 424 78.9 79.3 72.7 85.9 77.8 328123 425 120.8 106.7 85.4 162.4 74.7 328124 426 99.1 101.8 103.6 122.7 90.4 328125 427 81.7 86.9 75.8 99.5 76.1 328126 428 98.9 90.9 83.2 100.7 75.0 328127 429 74.5 86.9 89.7 80.8 77.6 328128 430 98.7 100.7 94.1 101.9 84.0 328129 431 53.8 67.6 56.5 60.0 71.8 328130 432 122.4 86.6 76.5 83.8 99.4 328131 433 89.1 95.4 81.8 103.6 88.2 328132 434 114.1 90.2 73.7 72.1 90.0 328133 435 61.2 69.5 63.0 91.9 63.8 328134 436 85.7 80.1 74.7 88.3 78.4 328135 437 63.6 80.6 76.7 90.3 70.0 328136 438 47.0 73.0 65.0 66.7 72.7 328137 439 83.2 99.6 86.3 88.5 85.7 328138 440 100.6 85.3 89.8 86.8 83.8 328139 441 89.1 98.3 92.6 106.3 115.0

TABLE 18 Effects of oligomeric compounds targeting miRNAs on expression of adipocyte differentiation markers SEQ ISIS # ID NO TG AP2 HSL Glut4 PPAR gamma Untreated N/A 102.2 90.8 94.9 117.8 103.3 control 105990 759 32.8 49.8 52.0 68.1 60.1 342673 758 100 100 100 100 100 TNF-alpha N/A 14.5 9.6 3.1 1.9 27.9 Undiff. N/A 2.8 0.0 1.4 0.3 10.7 adipocytes 327912 330 107.4 90.1 90.6 89.0 76.9 327969 387 46.0 59.8 66.4 60.6 69.2 328099 401 93.9 85.9 88.4 86.8 81.9 328100 402 71.5 61.9 72.0 74.2 66.7 328101 403 108.6 83.2 91.8 84.7 79.3 328102 404 95.9 87.9 97.0 79.2 93.7 328103 405 110.2 83.2 82.5 94.3 74.3 328104 406 122.6 102.2 98.2 119.1 90.4 328105 407 93.1 88.2 94.2 94.2 93.3 328106 408 90.5 88.8 94.9 105.7 90.7 328107 409 66.7 67.5 61.0 72.5 79.3 328108 410 89.6 83.7 90.1 94.9 84.0 328109 411 84.9 84.9 86.9 106.6 96.1 328110 412 97.7 93.3 91.0 104.7 91.2 328111 413 101.9 71.5 69.5 59.6 74.9 328112 414 98.1 99.1 101.2 122.5 102.4 328113 415 80.8 84.5 90.6 99.9 93.8 328114 416 117.3 94.4 93.3 114.9 89.3 328115 417 108.7 80.0 89.0 132.0 95.8 341803 760 85.9 77.3 75.5 86.8 71.2 341804 761 60.9 70.8 71.6 73.6 74.1 341805 762 78.1 81.9 81.8 88.2 80.4 341806 763 83.2 75.8 73.4 69.4 72.6 341807 764 114.1 74.8 96.8 119.5 86.2

Several compounds were found to have remarkable effects. For example, the oligomeric compounds ISIS Number 328117 (SEQ ID NO: 419), targeted to hypothetical miRNA-144, ISIS Number 328129 (SEQ ID NO: 431), targeted to hypothetical miRNA-173, ISIS Number 328136 (SEQ ID NO: 438), targeted to hypothetical miRNA-181, and ISIS Number 327969 (SEQ ID NO: 387), targeted to mir-182, were each shown to reduce the expression levels of triglycerides by at least 50%, and treatment with ISIS 328117, 328129, or 328136 also each resulted in a reduction of expression of the other four hallmark genes, indicating that these oligomeric compounds targeted to hypothetical miRNA-144, hypothetical miRNA-173, hypothetical miRNA-181, and mir-182, may be useful as therapeutic agents with applications in the treatment, attenuation or prevention of obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases.

The oligomeric compound ISIS Number 328121 (SEQ ID NO: 423), targeted to hypothetical miRNA-161 is an example of a compound which stimulates an increase in all five markers of adipocyte differentiation. This oligomeric compound may be useful as a pharmaceutical agent in the treatment of diseases in which the induction of adipocyte differentiation is desirable, such as anorexia, or for conditions or injuries in which the induction of cellular differentiation is desirable, such as Alzheimers disease or central nervous system injury, in which regeneration of neural tissue would be beneficial. Furthermore, this oligomeric compound may be useful in the treatment, attenuation or prevention of diseases in which it is desirable to induce cellular differentiation and/or quiescence, for example in the treatment of hyperproliferative disorders such as cancer.

Example 14 Expression of Mir-143 in Human Tissues and Cell Lines

Total RNA from spleen, kidney, testicle, heart and liver tissues as well as total RNA from human promyelocytic leukemia HL-60 cells, human embryonic kidney 293 (HEK293) cells, and T47D human breast carcinoma cells was purchased from Ambion, Inc. (Austin, Tex.). RNA from preadipocytes and differentiated adipocytes was purchased from Zen-Bio, Inc. (Research Triangle Park, N.C.). RNA was prepared from the HeLa, NT2, T-24, and A549 cell lines cultured as described above, using the following protocol: cell monolayers were washed twice with cold PBS, and cells were lysed in 1 mL TRIZOL™ (Invitrogen) and total RNA prepared using the manufacturer's recommended protocols.

Fifteen to twenty micrograms of total RNA was fractionated by electrophoresis through 10% acrylamide urea gels using a TBE buffer system (Invitrogen). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by electroblotting in an Xcell SureLock™ Minicell (Invitrogen). Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using Rapid Hyb buffer solution (Amersham) using manufacturer's recommendations for oligonucleotide probes.

To detect mir-143, a target specific DNA oligonucleotide probe with the sequence TGAGCTACAGTGCTTCATCTCA (SEQ ID NO: 319) was synthesized by IDT (Coralville, Iowa). The oligo probe was 5′ end-labeled with T4 polynucleotide kinase with (γ-³²P) ATP (Promega). To normalize for variations in loading and transfer efficiency membranes can be stripped and probed for U6 RNA. Hybridized membranes were visualized and quantitated using a Storm 860 PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.).

Using this probe, the mir-143 miRNA was found to be most highly expressed in human heart, thymus and kidney, and was also expressed to a lesser extent in lung, spleen, liver, and brain tissues. For example, as compared to expression levels in liver, mir-143 was expressed approximately 24-fold higher in heart, 17-fold higher in thymus, and 8-fold higher in kidney.

The mir-143 miRNA was also found to be expressed in adipocytes and preadipocytes, and levels of mir-143 were found to be dramatically upregulated in differentiated adipocytes as compared to preadipocytes, indicating that this miRNA may be important in adipocyte differentiation. These data, taken together with the finding that the oligomeric compound, ISIS Number 327901 (SEQ ID NO: 319), targeted to mir-143, was shown to inhibit the adipocyte differentiation markers (described above, Example 13), supports the conclusion that mir-143 is involved in cellular differentiation pathways.

Example 15 Effects of Oligomeric Compounds Targeting miRNAs on Apoptosis in the Caspase Assay in Preadipocytes

The effect of oligomeric compounds of the present invention targeting miRNAs was examined in preadipocytes (Zen-Bio, Inc., Research Triangle Park, N.C.) using the fluorometric caspase assay previously described in Example 11. The oligonucleotide random-mer, ISIS-29848 (SEQ ID NO: 737) was used as a negative control, and ISIS-148715 (SEQ ID NO: 738), targeting the human Jagged2 mRNA, known to induce apoptosis when inhibited, was used as a positive control. The measurement obtained from the untreated control cells is designated as 100% activity and was set equal to 1.0. Results are shown in Table 19.

TABLE 19 Effects of targeting miRNAs on apoptosis in preadipocytes SEQ ID Fold Increase ISIS Number NO. Pri-miRNA over UTC UTC N/A N/A 1.0 Untreated control ISIS-29848 737 N/A 1.2 n-mer ISIS-148715 738 Jagged2 36.9 Positive control 327888 306 mir-108-1 1.1 327889 307 mir-23b 1.1 327890 308 let-7i 1.3 327893 311 let-7b 1.3 327901 319 mir-143 2.0 327903 321 let-7a-3 1.6 327905 323 mir-205 1.5 327919 337 mir-221 1.3 327922 340 mir-19b-2 1.0 327925 343 mir-133b 2.0 327926 344 let-7d 1.8 327930 348 let-7e 1.4 327931 349 let-7c 1.5 327934 352 mir-213 2.0 327938 356 mir-98 1.0 327939 357 mir-125b-1 2.2 327941 359 mir-181b 1.3 327942 360 mir-141 1.0 327955 373 mir-130b 4.3 327967 385 let-7g 1.5

From these data, it is evident that the oligomeric compounds of the present invention generally do not induce the activity of caspases involved in apoptotic pathways in preadipocytes. In particular, the oligomeric compound targeting mir-143, ISIS Number 327901 (SEQ ID NO: 319), does not result in a significant increase in caspase activity as compared to the Jagged2 positive control. Taken together with the results from the adipocyte differentiation assay (Example 13) and the expression analysis of mir-143 (Example 14), these data suggest that the mir-143 miRNA plays a role in stimulating cellular differentiation, employing pathways other than the caspase cascades activated during apoptosis.

It was recently reported that bone marrow cells may contribute to the pathogenesis of vascular diseases, and that cell differentiation appears to be important in models of postangioplasty restenosis, graft vasculopathy, and hyperlipidemia-induced atherosclerosis. Bone marrow cells have the potential to give rise to vascular progenitor cells that home in on damaged vessels and differentiate into smooth muscle cells or endothelial cells, thereby contributing to vascular repair, remodeling, and lesion formation (Sata, M. Trends Cardiovasc Med. 2003 13(6):249-53). Thus, the ability to modulate cell differentiation may provide the basis for the development of new therapeutic strategies for vascular diseases, targeting mobilization, homing, differentiation, and proliferation of circulating vascular progenitor cells.

Example 16 Comparison of Effects of Oligomeric Compounds Targeting the Mir-143 Pri-miRNA or Mature Mir-143 miRNA on Adipocyte Differentiation

Two oligomeric compounds targeting the mature mir-143 miRNA and two oligomeric compounds targeting the 110-nucleotide mir-143 pri-miRNA were compared for their effects on adipocyte differentiation using the same adipocyte differentiation assay as described in Example 13.

The oligomeric compound, ISIS Number 327901 (SEQ ID NO: 319), 22-nucleotides in length, targets the mature mir-143 miRNA and is composed of 2′-methoxyethoxy (2′-MOE) nucleotides and phosphorothioate (P═S) internucleoside (backbone) linkages throughout. The oligomeric compound ISIS Number 338664 (CAGACTCCCAACTGACCAGA; SEQ ID NO: 491) is also a uniform 2′-MOE oligonucleotide, which is designed to target the mir-143 pri-miRNA. Another oligomeric compound targeting the mir-143 pri-miRNA, ISIS Number 328382 (SEQ ID NO: 491) is a chimeric oligonucleotide, 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings” having 2′-MOE substituents in the wing nucleosides (a “5-10-5 gapmer”), and ISIS Number 340927 (TGAGCTACAGTGCTTCATCTCA; SEQ ID NO: 319) is a 5-10-7 gapmer designed to target mature mir-143. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The effect of these oligomeric compounds targeting the mir-143 miRNA and the mir-143 pri-miRNA on expression of the 5 hallmark genes indicating cellular differentiation was examined in preadipocytes using the same methods described in Example 13. Results are shown in Table 20.

TABLE 20 Comparison of uniform 2′-MOE and chimeric oligomeric compounds targeting the mir-143 miRNA and pri-miRNAs on expression of adipocyte differentiation markers ISIS SEQ ID Number NO TG AP2 HSL Glut4 PPAR gamma 327901 319 0.54 0.42 0.33 0.19 0.30 328382 491 0.72 0.89 0.75 0.85 0.96 338664 491 1.42 1.01 0.76 1.81 0.86 340927 319 0.65 0.77 0.73 0.54 0.36

From these data, it was observed that while the gapmer oligomeric compound targeting the mature mir-143 (ISIS Number 340972) results in reduced expression of the adipocyte differentiation markers, the uniform 2′-MOE oligomeric compound targeting mature mir-143 (ISIS Number 327901) was more effective. For the oligomeric compounds targeting the mir-143 pri-miRNA, the gapmer compound (ISIS Number 328382) appeared to be more effective in blocking adipocyte differentiation than was the uniform 2′-MOE oligomeric compound (ISIS Number 338664).

Dose Responsiveness:

In one embodiment, the oligomeric compound ISIS Number 327901 (SEQ ID NO: 319) targeting mature mir-143 was selected for additional dose response studies in the adipocyte differentiation assay. Differentiating adipocytes (at day 10 post-induction of differentiation) were treated with 50, 100, 200, and 300 nM ISIS 327901, or the scrambled control ISIS Number 342673 (SEQ ID NO: 758) containing 15 mismatches with respect to the mature mir-143 miRNA. ISIS Numbers 327901 and 342673 are uniform 2′-MOE phosphorothioate oligomeric compounds 22 nucleotides in length. Differentiating adipocytes treated with ISIS Number 29848 (SEQ ID NO: 737) served as the negative control to which the data were normalized. Differentiating adipocytes treated with ISIS 105990 (SEQ ID NO: 759), a 5-10-5 gapmer oligomeric compound targeting the PPAR-gamma mRNA which has been demonstrated previously to inhibit adipocyte differentiation, served as the positive control. Triglyceride levels as well as mRNA levels for each of the four adipocyte differentiation hallmark genes (PPAR-gamma, aP2, HSL, and GLUT4) were measured 24 hours after treatment as described above. Untreated cells were compared to cells treated with oligomeric compounds, and results of these dose response studies are shown in Table 21, where levels of the markers is expressed as a percentage of untreated control (% UTC) levels. Where present, “N.D.” indicates “no data.”

TABLE 21 Effects of oligomeric compounds targeting mir-143 on expression of adipocyte differentiation markers % UTC Hallmark Dose of oligomeric compound Measured: Isis #: 50 nM 100 nM 200 nM 300 nM Triglycerides 342673 94.2 105.3  98.3 108.2 negative control 105990 N.D. N.D. N.D. 16.6 positive control 327901 85.3 68.9 34.0 23.0 PPAR-gamma 342673 77.5 89.9 94.6 85.8 mRNA negative control 105990 N.D. N.D. N.D. 43.9 positive control 327901 74.6 70.8 51.8 39.3 AP2 mRNA 342673 82.4 90.3 81.1 70.9 negative control 105990 N.D. N.D. N.D. 17.9 positive control 327901 78.3 64.6 39.0 22.4 HSL mRNA 342673 92.0 95.6 97.3 85.2 negative control 105990 N.D. N.D. N.D. 7.4 positive control 327901 89.5 73.5 40.2 11.9 GLUT4 mRNA 342673 94.9 90.7 97.6 102.7 negative control 105990 N.D. N.D. N.D. 11.8 positive control 327901 74.2 49.7 32.8 17.4

From these data, it was observed that treatment of differentiating adipocytes with the uniform 2′-MOE oligomeric compound, ISIS Number 327901 targeting mir-143 results in a dose responsive reduction of expression of all five markers of differentiation. Thus, this oligomeric compound may be useful in the treatment of diseases associated with increased expression of these hallmark genes, such as obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases as well as having potential applications in the maintenance of the pluripotent phenotype of stem or precursor cells.

Example 17 Human Let 7 Homologs

Let-7 is one of the two miRNAs originally identified in C. elegans as an antisense translational repressor of messenger RNAs encoding key developmental timing regulators in nematode larva. Several genes predicted to encode let-7-like miRNAs have been identified in a wide variety of species, and these let-7-like homologs are believed to control temporal transitions during development across animal phylogeny. Oligomeric compounds of the present invention were designed to target several human let-7-like genes. Additionally, a series of target-specific DNA oligonucleotide probes were synthesized by IDT (Coralville, Iowa) and used in Northern analyses to assess the expression of let-7-like miRNA homologs in various tissues. These let-7 homolog specific probes are shown in Table 22.

TABLE 22 Probes for Northern analyses of mRNA expression of let-7 homologs ISIS Number SEQ ID NO Sequence pri-miRNA 327890 308 AGCACAAACTACTACCTCA let-7i 327893 311 AACCACACAACCTACTACCTCA let-7b 327903 321 AACTATACAACCTACTACCTCA let-7a-3 327926 344 ACTATGCAACCTACTACCTCT let-7d 327930 348 ACTATACAACCTCCTACCTCA let-7e 327931 349 AACCATACAACCTACTACCTCA let-7c 327967 385 ACTGTACAAACTACTACCTCA let-7g

For Northern analyses with let-7 homolog probes, total RNA from spleen, kidney, testes, heart, and liver tissues as well as total RNA from HEK293, T47D, T-24, MCF7, HepG2, and K-562 Leukemia cell lines was either prepared as described above or purchased from Ambion, Inc. (Austin, Tex.). Northern blotting was performed as described above (Example 14). The let-7c miRNA was observed to be expressed in spleen, kidney, testes, heart and liver tissues, as well as in HEK293 and T47D cell lines. The let-7e miRNA was observed to be expressed in T-24, MCF7, T47D, 293T, HepG2, and K-562 cell lines.

In one embodiment, expression of let-7-like pri-miRNA homologs was detected in total RNA from brain, liver and spleen tissues, as well as total RNA from preadipocytes, differentiated adipocytes, and HeLa, HEK-293, and T-24 cell lines by real-time RT-PCR. Primer/probe sets were designed to distinguish between and amplify specific let-7-like pri-miRNA homologs. These primer/probe sets are shown in Table 23.

TABLE 23 Primer/probe sets for assaying expression of let-7 miRNA homologs Primer or Isis SEQ ID Pri-miRNA probe number NO. sequence let-7b forward 341672 765 GAGGTAGTAGGTTGTGTGGTTTCA reverse 341673 766 AGGGAAGGCAGTAGGTTGTATAGTT probe 341674 767 CAGTGATGTTGCCCCTCGGAAGA let-7c forward 341675 768 TGCATCCGGGTTGAGGTA reverse 341676 769 AGGAAAGCTAGAAGGTTGTACAGTTAA probe 341677 770 AGGTTGTATGGTTTAGAGTTACACCCTGGGA let-7d forward 341678 771 CCTAGGAAGAGGTAGTAGGTTGCA reverse 341679 772 CAGCAGGTCGTATAGTTACCTCCTT probe 341680 773 AGTTTTAGGGCAGGGATTTTGCCCA let-7g forward 341681 774 TTCCAGGCTGAGGTAGTAGTTTG reverse 341682 775 TTATCTCCTGTACCGGGTGGT probe 341683 776 ACAGTTTGAGGGTCTAT let-7i forward 341684 777 TGAGGTAGTAGTTTGTGCTGTTGGT reverse 341685 778 AGGCAGTAGCTTGCGCAGTTA probe 341686 779 TTGTGACATTGCCCGCTGTGGAG let-7a-1 forward 341687 780 GGATGAGGTAGTAGGTTGTATAGTTTTAGG reverse 341688 781 CGTTAGGAAAGACAGTAGATTGTATAGTTATC probe 341689 782 TCACACCCACCACTGG let-7a-3 forward 341690 783 GGGTGAGGTAGTAGGTTGTATAGTTTGG reverse 341691 784 CACTTCAGGAAAGACAGTAGATTGTATAGTT probe 341692 785 CTCTGCCCTGCTATGG

Using these primer/probe sets, the let-7-like pri-miRNA homologs were found to be expressed in human brain, liver and spleen, as well as preadipocytes, differentiated adipocytes, and HeLa, T-24 and HEK-293 cells lines. In particular, the let-7b pri-miRNA exhibited approximately 100-fold higher expression in differentiated adipocytes as compared to preadipocytes. Furthermore, the let-7b, let-7c, let-7d, let-71, and let-7a-3 pri-miRNAs were highly expressed in brain and spleen tissues.

In summary, the let-7-like homologs have been found to be widely expressed in various human tissues and several cell lines. Furthermore, some oligomeric compounds targeted to human let-7 pri-miRNAs generally appeared to result in the induction of cell differentiation, consistent with the functional role of let-7 as a regulator of developmental timing in nematode larva. Specifically, the oligomeric compounds targeted to let-7c (ISIS Number 327931; SEQ ID NO: 349) and let-7a-3 (ISIS Number 327903; SEQ ID NO: 321) resulted in an increase in expression levels for several markers of adipocyte differentiation. Furthermore, inhibition of the let-7-like homologs by oligomeric compounds of the present invention did not appear to induce caspases activated in apoptotic pathways (performed in Example 15). Thus, the oligomeric compounds of the present invention targeting let-7-like pri-miRNA homologs appear to stimulate adipocyte differentiation and do not promote cell death by apoptosis. Thus, the oligomeric compounds of the present invention may be useful as pharmaceutical agents in the treatment of anorexia or diseases, conditions or injuries in which the induction of cellular differentiation is desirable, such as Alzheimers disease or central nervous system injury, in which neural regeneration would be beneficial.

Example 18 Effects of Oligomeric Compounds Targeting miRNAs on Insulin Signaling in HepG2 Cells

Insulin is secreted from pancreatic β-cells in response to increasing blood glucose levels. Through the regulation of protein expression, localization and activity, insulin ultimately stimulates conversion of excess glucose to glycogen, and results in the restoration of blood glucose levels. Insulin is known to regulate the expression of over 100 gene products in multiple cell types. For example, insulin completely inhibits the expression of hepatic insulin-like growth factor binding protein-1 (IGFBP-1), a protein which can sequester insulin-like growth factors, and phosphoenolpyruvate carboxykinase-cytosolic (PEPCK-c) which is a rate-controlling enzyme of hepatic gluconeogenesis. Levels of the follistatin mRNA are also believed to decrease in response to insulin treatment. IGFBP-1 and PEPCK-c are overexpressed in diabetes, and PEPCK-c overexpression in animals promotes hyperglycemia, impaired glucose tolerance and insulin-resistance. Thus, the IGFBP-1, PEPCK-c and follistatin genes serve as marker genes for which mRNA expression can be monitored and used as an indicator of an insulin-resistant state. Oligomeric compounds with the ability to reduce expression of IGFBP-1, PEPCK-c and follistatin are highly desirable as agents potentially useful in the treatment of diabetes and hypertension.

Oligomeric compounds of the present invention were tested for their effects on insulin signaling in HepG2 cells. HepG2 cells were plated at 7500 cells/well in collagen coated 96-well plates. The following day, cells were transfected with oligomeric compounds targeting miRNAs using 100 nM oligomeric compound in LIPOFECTIN™ (Invitrogen Corporation, Carlsbad, Calif.) in two 96-well plates. The oligomeric compounds were tested in triplicate on each 96-well plate, except for positive and negative controls, which were measured up to six times per plate. At the end of transfection, the transfection medium was replaced by regular growth medium. Twenty-eight hours post-transfection, the cells were subjected to overnight (sixteen to eighteen hours) serum starvation using serum free growth medium. Forty-four hours post-transfection, the cells in the transfected wells were treated with either no insulin (“basal” Experiment 1, for identification of insulin-mimetic compounds) or with 1 nM insulin (“insulin treated” Experiment 2, for identification of insulin sensitizers) for four hours. At the same time, in both plates, cells in some of the un-transfected control wells are treated with 100 nM insulin to determine maximal insulin response. At the end of the insulin or no-insulin treatment (forty-eight hours post-transfection), total RNA is isolated from both the basal and insulin treated (1 nM) 96-well plates, and the amount of total RNA from each sample is determined using a Ribogreen assay (Molecular Probes, Eugene, Oreg.). Real-time PCR is performed on all the total RNA samples using primer/probe sets for three insulin responsive genes: PEPCK-c, IGFBP-1 and follistatin. Expression levels for each gene are normalized to total RNA, and values±standard deviation are expressed relative to the transfectant only untreated control (UTC) and negative control compounds. Results of these experiments are shown in Tables 24 and 25.

TABLE 24 Experiment 1: Effects of oligomeric compounds targeting miRNAs on insulin-repressed gene expression in HepG2 cells ISIS SEQ ID IGFBP-1 PEPCK-c Follistatin Number NO Pri-miRNA (% UTC) (% UTC) (% UTC) UTC N/A N/A 100 100 100  29848 737 N/A 95 87 94 n-mer 327876 294 mir-29b-1 93 119 104 327878 296 mir-203 162 45 124 327880 298 mir-10b 137 110 107 327889 307 mir-23b 56 137 56 327890 308 let-7I 99 85 78 327892 310 mir-131-2/mir-9 108 75 91 327901 319 mir-143 133 119 93 327903 321 let-7a-3 71 71 60 327905 323 mir-205 107 129 104 327913 331 mir-29c 123 229 115 327919 337 mir-221 96 71 74 327922 340 mir-19b-2 109 77 57 327925 343 mir-133b 152 145 110 327933 351 mir-145 125 118 112 327934 352 mir-213 231 99 140 327939 357 mir-125b-1 125 125 104 327941 359 mir-181b 83 101 80 327954 372 mir-148b 118 79 100 338664 491 mir-143 pri- 90 75 93 miRNA 340927 319 mir-143 201 87 111

Under “basal” conditions (without insulin), treatments of HepG2 cells with oligomeric compounds of the present invention resulting in decreased mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that the oligomeric compounds have an insulin mimetic effect. Treatments with oligomeric compounds of the present invention resulting in an increase in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds inhibit or counteract the normal insulin repression of mRNA expression of these genes.

From these data, it is evident that the oligomeric compounds, ISIS Number 327878 targeting mir-203 and ISIS Number 327922 targeting mir-19b-2, for example, result in a 55% and a 23% decrease, respectively, in PEPCK-c mRNA, a marker widely considered to be insulin-responsive. Thus, these oligomeric compounds may be useful as pharmaceutic agents comprising insulin mimetic properties in the treatment, amelioration, or prevention of diabetes or other metabolic diseases.

Conversely, the results observed with the oligomeric compound targeting mir-29c (ISIS Number 327913), for example, exhibiting increased expression of the IGFBP-1, PEPCK-c and follistatin marker genes, suggest that the mir-29c miRNA target may be involved in the regulation of these insulin-responsive genes. When the mir-29c miRNA is inactivated by an oligomeric compound, IGFBP-1, PEPCK-c and follistatin gene expression is no longer repressed.

TABLE 25 Experiment 2: Effects of oligomeric compounds targeting miRNAs on insulin-sensitization of gene expression in HepG2 cells ISIS SEQ ID IGFBP-1 PEPCK-c Follistatin Number NO Pri-miRNA (% UTC) (% UTC) (% UTC) UTC N/A N/A 100 100 100 (1 nm insulin)  29848 737 N/A 92 94 97 n-mer 327876 294 mir-29b-1 118 176 138 327878 296 mir-203 185 29 150 327880 298 mir-10b 136 125 149 327890 307 let-7i 88 113 115 327892 308 mir-131-2/mir-9 139 104 96 327901 310 mir-143 135 117 135 327903 319 let-7a-3 81 87 89 327905 321 mir-205 115 147 148 327913 323 mir-29c 147 268 123 327919 331 mir-221 154 105 178 327922 337 mir-19b-2 104 76 61 327925 340 mir-133b 166 182 148 327933 343 mir-145 179 115 185 327934 351 mir-213 244 105 103 327939 352 mir-125b-1 175 153 192 327941 357 mir-181b 80 98 68 327954 359 mir-148b 120 102 105 327889 372 mir-23b 73 202 72 338664 491 mir-143 pri- 100 76 84 miRNA 340927 319 mir-143 285 103 128

For HepG2 cells treated with 1 nM insulin, treatments with oligomeric compounds of the present invention resulting in a decrease in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds have an insulin sensitization effect. Treatments with oligomeric compounds of the present invention resulting in an increase in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds inhibit or counteract the normal insulin response of repression of mRNA expression of these genes.

From these data, it is evident that the oligomeric compounds, ISIS Number 327878 targeting mir-203 and ISIS Number 327922 targeting mir-19b-2, for example, were observed to result in a 71% and a 24% reduction, respectively, of PEPCK-c mRNA expression, widely considered to be a marker of insulin-responsiveness. Thus, these oligomeric compounds may be useful as pharmaceutic agents with insulin-sensitizing properties in the treatment, amelioration, or prevention of diabetes or other metabolic diseases.

Conversely, the results observed with the oligomeric compounds targeting mir-29c (ISIS Number 327913), mir-133b (ISIS Number 327925), and mir-125b-1 (ISIS Number 327939), all exhibiting increased expression of the IGFBP-1, PEPCK-c and follistatin marker genes, support the conclusion that the mir-29c, mir-133b, and mir-125b-1 miRNAs may be involved in the regulation of insulin-responsive genes. When these miRNAs are inactivated by the oligomeric compounds of the present invention, IGFBP-1, PEPCK-c and follistatin gene expression is no longer repressed or insulin-sensitive.

A caspase assay was also performed (as in Example 11 above) in HepG2 cells treated with oligomeric compounds of the present invention, and it was determined that oligomeric compounds targeting the mir-29c, mir-133b, and mir-125b-1 miRNAs were not toxic to the cells and that the observed reduction in mRNA expression levels of insulin-responsive genes was not due to a general toxicity of the compounds or an induction of apoptotic pathways.

Example 19 Analysis of Expression of Mir-143 Pri-miRNA and Mature Mir-143

Ribonuclease Protection Assays:

The ribonuclease protection assay (RPA) is known in the art to be a sensitive and accurate method of measuring and/or following temporal changes in the expression of one or more RNA transcripts in a complex mixture of total RNA. Briefly, this method employs a radioactive probe that specifically hybridizes to a target transcript RNA. The probe is added to a sample of total RNA isolated from tissues or cells of interest, and, upon hybridization to its target, the probe forms a double-stranded RNA region. If the region of hybridization is shorter than the entire length of either the probe or the target RNA molecule, the molecule will be a hybrid molecule with partial double-stranded and partial single-stranded character. These hybrid molecules are then digested with single-strand-specific RNases such as RNase A and/or T1, which remove any non-hybridized single stranded portions of the hybrid molecules, leaving only the “protected” dsRNA fragments. The RNase protected fragments are then electrophoresed on a denaturing gel, causing the strands to dissociate, and the intensity of radioactive probe signal observed is directly proportional to the amount of specific target transcript RNA in the original total RNA sample.

In an embodiment of the present invention, small non-coding RNAs in a sample were detected by RPA using probes that hybridize to pri-miRNAs, pre-miRNAs or mature miRNAs. Probes were in vitro transcribed using the mirVana™ miRNA Probe Construction Kit (Ambion Inc., Austin, Tex.) according to the manufacturer's protocol, beginning with a DNA oligonucleotide representing sense strand of the mature miRNA to be detected plus four thymidylate residues plus an 8-base sequence complementary to the 3′-end of the T7 promoter primer supplied with the kit. When the T7 primer is annealed to this DNA oligonucleotide, the Klenow DNA polymerase is used to generate a double-stranded DNA, and then in vitro transcription is performed using the T7 RNA polymerase and radiolabeled nucleotides to generate a radioactive RNA probe for detection of the miRNA.

In one embodiment, a probe specifically hybridizing to the murine mir-143 miRNA was used in a RPA of 5 μg total RNA from kidney, liver, heart, lung, brain, spleen, and thymus tissues from mouse as well as adipose tissue from db/db obese mice, total RNA from an 11-day-old embryo, and total RNA from undifferentiated and differentiated 3T3-L1 cells. All signals were normalized to the levels of 5.8S rRNA. Expression levels of mir-143 were highest in lung, heart, spleen, thymus and kidney tissues from wildtype mice. Notably, mir-143 expression levels were significantly elevated in adipose tissue from db/db mice (approximately 4 times higher than expression levels in kidney, 2.4 times higher than levels in heart and 1.6 times higher than levels in lung tissues from wildtype mice).

In one embodiment, a probe hybridizing to the mir-143 pri-miRNA molecule was used in a RPA of 2-5 μg total RNA from human spleen, thymus, testes, heart, liver, kidney, skeletal muscle, brain, lung and adipose tissues, as well as total RNA from preadipocytes, differentiated adipocytes, and HepG2 cells. A probe hybridizing to the β-actin mRNA was used as a control. The highest levels of mir-143 pri-miRNA were observed in heart, kidney, thymus and adipose tissues, as well as in differentiated adipocytes.

In one embodiment, a probe hybridizing to the mature mir-143 miRNA was also used in a RPA of 2 μg total RNA from human spleen, thymus, heart, liver, kidney and brain, tissues, as well as total RNA from preadipocytes, differentiated adipocytes, and total RNA from HepG2, A549, T-24, HEK293, HuVEC (human umblical vein endothelial cells), HL-60 and T47D cell lines. A probe hybridizing to the β-actin mRNA was used as a control, and all signals were normalized to the levels of mir-143 expression in preadipocytes. The results are shown in Table 26.

TABLE 26 RNase protection of mature mir-143 in total RNA from tissues and cell lines Tissue or cell line Fold Increase over preadipocytes Spleen 2.6 Thymus 3.8 Heart 8.2 Liver 0 Kidney 10.0 Brain 0.9 Preadipocytes 1.0 Differentiated 2.6 adipocytes HepG2 0.5 A549 N.D. T-24 0.4 HEK293 0.5 HuVEC 0.3 HL-60 0.4 T47D 0.3

From these data, the highest levels of expression of the mature mir-143 miRNA were observed in total RNA from kidney and heart tissues. High levels of expression of the mature mir-143 miRNA were also observed in total RNA from lymphoid tissues such as spleen and thymus. Expression of the mature mir-143 miRNA is increased in differentiated adipocytes as compared to levels in preadipocytes. These data also suggest that the mir-143 miRNA plays a role in cellular differentiation.

In one embodiment, a uniform 2′-MOE phosphorothioate oligomeric compound with a sequence antisense to the mature mir-143 miRNA was spiked into the RPA mixture above. This antisense mir-143 compound was found to block the ribonuclease protection expression pattern previously observed, suggesting that this antisense mir-143 oligomeric compound specifically hybridizes to and inhibits the activity of mir-143. This oligomeric compound targeting the mir-143 miRNA is predicted to form a double stranded molecule that blocks endogenous mir-143 miRNA activity when employed in vivo.

It was also noted that, while expression of the mir-143 miRNA can be detected in non-transformed cells, such as HuVECs, in general, transformed cell lines have not been observed to exhibit high levels expression of mir-143. When taken together with the observation that the mir-143 miRNA is upregulated as adipocytes differentiate as well as the observation that oligomeric compounds targeting mir-143 inhibit adipocyte differentiation, these data suggest that mir-143 normally promotes adipocyte differentiation and mir-143 may have an inhibitory effect on cellular transformation that is consistent with its role in promoting cellular differentiation. Lack of expression or downregulation of mir-143 in transformed cell lines may be a cause or consequence of the undifferentiated state. Thus, mir-143 mimics may be useful as pharmaceutical agents in the treatment of hyperproliferative disorders such as cancer.

In one embodiment, the expression of human mir-143 was assessed during adipocyte differentiation. A probe hybridizing to the human mir-143 miRNA was used in a RPA of 5 μg total RNA from pre-adipocytes, and differentiated adipocytes sampled at one, four, and ten days post-differentiation. All signals were normalized to the levels of 5.8S rRNA. mir-143 expression levels were 2.5 to 3-fold higher by day 10 post-differentiation when compared to mir-143 expression levels in pre-adipocytes by ribonuclease protection assay.

Real-Time RT-PCR Analysis of mir-143 Pri-miRNA Expression:

Expression levels of mir-143 pri-miRNA were compared in total RNAs from various tissues and total RNA from several cell lines. Total RNA from spleen, heart, liver, and brain tissues, as well as total RNA from preadipocytes, differentiated adipocytes, and HepG2, T-24 and HeLa cell lines was purchased or prepared as described supra. 80 ng of total RNA from each source was used to perform real-time RT-PCR using a primer/probe set specific for the mir-143 pri-miRNA molecule. ISIS 339314 (TCCCAGCCTGAGGTGCA; SEQ ID NO: 786) was used as the forward primer, ISIS 342897 (GCTTCATCTCAGACTCCCAACTG; SEQ ID NO: 787) was used as the reverse primer, and ISIS 342898 (TGCTGCATCTCTG; SEQ ID NO: 788) was used as the probe. RNA levels from all sources were compared to RNA levels from preadipocytes. Greater than 32-fold higher levels of mir-143 pri-miRNA were observed in heart tissue as compared to preadipocytes; 19-fold higher levels of mir-143 pri-miRNA were observed in differentiated adipocytes relative to levels in preadipocytes; 5-fold higher levels of mir-143 pri-miRNA were observed in spleen as compared to preadipocytes.

Northern blot analyses were performed in differentiating adipocytes as described in Example 14 using the mir-143-specific DNA oligonucleotide probe (SEQ ID NO: 319) to detect the mir-143 target and a probe for the U6 RNA to normalize for variations in loading and transfer efficiency, and it was confirmed by Northern analysis that expression of mature mir-143 increases from day 1 through day 10 after induction of differentiation.

In human pre-adipocytes and adipocytes sampled one, four, seven and ten days post-differentiation, expression levels of mir-143 pri-miRNA were also assessed using real-time RT-PCR analysis as described herein. 80 ng of total RNA from pre-adipocytes or differentiated adipocytes was used to perform real-time RT-PCR using the same primer/probe set specific for the mir-143 pri-miRNA molecule described supra (ISIS 339314, SEQ ID NO: 786 was used as the forward primer, ISIS 342897, SEQ ID NO: 787 was used as the reverse primer, and ISIS 342898, SEQ ID NO: 788 was used as the probe). RNA levels from all sources were normalized to 5.8S rRNA levels. mir-143 pri-miRNA levels in preadipocytes were 94% of the level of the 5.8S rRNA. At day 1 post-differentiation, mir-143 pri-miRNA levels had decreased to 38% of the level of the 5.8S rRNA. By day 4 post-differentiation, mir-143 pri-miRNA levels had decreased to 26%, by day 7 post-differentiation, mir-143 pri-miRNA levels were at 25%, and by day 10 post-differentiation, mir-143 pri-miRNA levels had dropped to 23% of the level of the 5.8S rRNA. Taken together with the results from RPA analysis, it appears that levels of the mature mir-143 miRNA increases approximately 2- to 3-fold by day 10 post-differentiation in differentiated adipocytes, accompanied by a concomitant approximately 4-fold decrease in the levels of unprocessed mir-143 pri-miRNA, indicating that adipocyte differentiation coincides with either an increase in processing of the mir-143 miRNA from the mir-143 pri-miRNA or an overall decrease in mir-143 pri-miRNA production.

Effects of Oligomeric Compounds on Expression of Pri-miRNAs:

Mature miRNAs originate from long endogenous primary transcripts (pri-miRNAs) that are often hundreds of nucleotides in length. It is believed that a nuclear enzyme in the RNase III family, known as Drosha, processes pri-miRNAs (which can range in size from about 110 nucleotides up to about 450 nucleotides in length) into pre-miRNAs (from about 70 to 110 nucleotides in length) which are subsequently exported from the nucleus to the cytoplasm, where the pre-miRNAs are processed by human Dicer into double-stranded intermediates resembling siRNAs, which are then processed into mature miRNAs. Using the real-time RT-PCR methods described herein, the expression levels of several pri-miRNAs were compared in differentiating adipocytes. Total RNA from preadipocytes and differentiating adipocytes was prepared as described herein.

In one embodiment, modified oligomeric compounds can be transfected into preadipocytes or other undifferentiated cells, which are then induced to differentiate (as described in detail, herein), and it can be determined whether these modified oligomeric compounds act to inhibit or promote cellular differentiation. Real-time RT-PCR methods can then be used to determine whether modified oligomeric compounds targeting miRNAs can affect the expression or processing of the pre-miRNAs from the pri-miRNA (by the Drosha enzyme), the processing of the mature miRNAs from the pre-miRNA molecules (by the Dicer enzyme), or the RISC-mediated binding of a miRNA to its target nucleic acid.

Here, oligomeric compounds targeting mir-143 were transfected into preadipocytes which were then induced to differentiate, in order to assess the effects of these compounds on mir-143 pri-miRNA levels during differentiation. mir-143 pri-miRNA levels were assessed on days 3 and 9 after differentiation.

In addition to the uniform 2′-MOE phosphorothioate oligomeric compound ISIS Number 327901 (SEQ ID NO: 319) targeting mature mir-143, a 5-10-7 gapmer oligomeric compound, ISIS Number 340927 (SEQ ID NO: 319), was designed to target mature mir-143. As negative controls, “scrambled” oligomeric compounds were also designed; ISIS Number 342672 (ATACCGCGATCAGTGCATCTTT; incorporated herein as SEQ ID NO: 789) contains 13 mismatches with respect to the mature mir-143 miRNA, and ISIS Number 342673 (SEQ ID NO: 758) contains 15 mismatches with respect to the mature mir-143 miRNA. ISIS 342672 and ISIS 342673 are uniform 2′-MOE phosphorothioate oligomeric compounds 22 nucleotides in length. ISIS Number 342677 (SEQ ID NO: 789) and ISIS Number 342678 (SEQ ID NO: 758) are the corresponding 5-10-7 scrambled 2′-MOE gapmer oligomeric compounds. All cytidine residues are 5-methylcytidines. Additionally, ISIS Number 342683 (CCTTCCCTGAAGGTTCCTCCTT; herein incorporated as SEQ ID NO: 790), representing the scrambled sequence of an unrelated PTP1B antisense oligonucleotide, was also used as a negative control.

These compounds were transfected into differentiating adipocytes and their effects on levels of the mir-143 pri-miRNA molecule were assessed in pre-adipocytes vs. differentiated adipocytes, by real-time RT-PCR using the primer/probe set specific for the mir-143 pri-miRNA (forward primer=ISIS 339314, SEQ ID NO: 786; reverse primer=ISIS 342897, SEQ ID NO.: 787; probe=ISIS 342898, SEQ ID NO.: 788). Thus, it was observed that in the presence of the oligomeric compound ISIS Number 327901 (SEQ ID NO: 319), levels of the mir-143 pri-miRNA are enhanced approximately 4-fold in differentiated adipocytes 9 days post-differentiation as compared to 3 days post-differentiation. These results suggest that ISIS Number 327901, the uniform 2′-MOE P═S oligomeric compound targeted to mature mir-143, interferes with the processing of the mir-143 pri-miRNA into the pre-miRNA by the Drosha RNase III enzyme. Alternatively, the compound interferes with the processing of the mir-143 pre-miRNA into the mature mir-143 miRNA by the Dicer enzyme. The decrease in levels of mature mir-143 miRNA in differentiating cells treated with ISIS Number 327901 (SEQ ID NO: 319) may also trigger a feedback mechanism that signals these cells to increase production of the mir-143 pri-miRNA molecule. Not mutually exclusive with the processing interference or the feedback mechanisms is the possibility that treatment with oligomeric compounds could stimulate the activity of an RNA-dependent RNA polymerase (RdRP) that amplifies the mir-143 pri-miRNA or pre-miRNA molecules. Oligomeric compounds of the present invention are predicted to disrupt pri-miRNA and/or pre-miRNA structures, and sterically hinder Drosha and/or Dicer cleavage, respectively. Furthermore, oligomeric compounds which are capable of binding to the mature miRNA are also predicted to prevent the RISC-mediated binding of a miRNA to its target nucleic acid, either by cleavage or steric occlusion of the miRNA.

Example 20 Identification of RNA Transcripts Bound by miRNAs

The RACE-PCR method (Rapid Amplification of cDNA Ends) was used as a means of identifying candidate RNA transcripts bound and/or potentially regulated by miRNAs. RNA was prepared and isolated from preadipocytes, and, using the SMART RACE cDNA Amplification kit (BD Biosciences, Clontech, Palo Alto, Calif.) according to manufacturer's protocol, synthetic adaptor sequences were incorporated into both the 5′- and 3′-ends of the amplified cDNAs during first strand cDNA synthesis. 5′ RACE-PCR was then performed using the mature miRNA as the 3′-end primer along with the 5′ adapter primer from the kit to amplify the 5′-end of the candidate RNA transcript. 3′ RACE-PCR was performed using the antisense sequence of the miRNA as a primer along with the 3′ adapter primer from the kit to amplify the 3′-end of the candidate RNA transcript. In some embodiments, the primers 2-nucleotides shorter than the corresponding miRNA were used in order to identify targets with some mismatching nucleotides at the end of the miRNA (these primers are indicated by “3′-RACE-2 nt” in Table 27 below).

For example, the antisense sequences of the mature mir-43, let-7g, mir-23b, mir-29c, mir-131, mir-143, mir-130b and mir-213 miRNAs were used as primers in 3′ RACE-PCR, and the mature mir-143 or mir-15a sequences were used in 5′ RACE-PCR. The RACE-PCR products employing the mir-143 miRNA, the mir-143 antisense sequence, the mir-131 antisense sequence or the mir-15a miRNA as primers were electrophoresed and gel purified, prominent bands were excised from the gel, and these products were subcloned using standard laboratory methods. The subcloned products from the RACE-PCR were then were sent to Retrogen, Inc. (San Diego, Calif.) for sequencing. Candidate RNA transcripts targeted by miRNAs were thereby identified.

Candidate RNA targets identified by RACE-PCR methods are shown in Table 27, where the miRNA-specific primer used to identify each transcript is indicated in the column entitled “primer”. (In some cases, the target was identified multiple times by more than one RACE-PCR method, and thus appears in the table more than once).

TABLE 27 Predicted RNA targets of mir-143 SEQ GenBank ID Primer Method Accession RNA transcript targeted by miRNA NO mir-143 5′RACE NM_001753.2 caveolin 1, caveolae protein, 22 kDa 791 mir-143 5′RACE NM_004652.1 ubiquitin specific protease 9, X- 792 linked (fat facets-like, Drosophila) mir-143 5′RACE NM_007126.2 valosin-containing protein 793 mir-143 5′RACE NM_000031.1 aminolevulinate, delta-, dehydratase 794 mir-143 5′RACE NM_007158.1 NRAS-related gene 795 mir-143 5′RACE NM_015396.1 HSPC056 protein 796 mir-143 5′RACE NM_001219.2 calumenin 797 mir-143 5′RACE BC051889.1 RNA binding motif, single stranded 798 interacting protein 1 mir-143 5′RACE BX647603.1 Homo sapiens mRNA; cDNA 799 DKFZp686L01105 (from clone DKFZp686L01105) mir-143 5′RACE AB051447.1 KIAA1660 protein 800 mir-143 5′RACE NM_007222.1 zinc-fingers and homeoboxes 1 801 mir-143 5′RACE NM_001855.1 collagen, type XV, alpha 1 802 mir-143 3′RACE NM_007222.1 zinc-fingers and homeoboxes 1 801 mir-143 3′RACE NM_006732 FBJ murine osteosarcoma viral 803 oncogene homolog B mir-143 3′RACE NM_003718.2 cell division cycle 2-like 5 804 (cholinesterase-related cell division controller) mir-143 3′RACE NM_005626.3 splicing factor, arginine/serine- 805 rich 4 mir-143 3′RACE NM_002355.1 mannose-6-phosphate receptor (cation 806 dependent) mir-143 3′RACE NM_000100.1 cystatin B (stefin B) 807 mir-143 3′RACE NM_015959.1 CGI-31 protein 808 mir-143 3′RACE NM_006769.2 LIM domain only 4 809 mir-143 3′RACE NM_003184.1 TAF2 RNA polymerase II, TATA box 810 binding protein (TBP)-associated factor, 150 kDa mir-143 3′RACE NM_025107.1 myc target in myeloid cells 1 811 mir-143 3′RACE NM_003113.1 nuclear antigen Sp100 812 mir-143 3′RACE NM_002696.1 polymerase (RNA) II (DNA directed) 813 polypeptide G mir-143 3′RACE NM_004156.1 protein phosphatase 2 (formerly 2A), 814 catalytic subunit, beta isoform mir-143 3′RACE NM_031157 heterogeneous nuclear 815 ribonucleoprotein A1 mir-143 3′RACE NM_004999.1 myosin VI 817 mir-143 3′RACE NM_018036.1 chromosome 14 open reading frame 103 818 mir-143 3′RACE NM_018312.2 chromosome 11 open reading frame 23 819 mir-143 3′RACE NM_002950.1 ribophorin I 820 mir-143 3′RACE NM_006708.1 glyoxalase I 821 mir-143 3′RACE NM_014953.1 mitotic control protein dis3 homolog 822 mir-143 3′RACE NM_004926.1 zinc finger protein 36, C3H type- 823 like 1 mir-143 3′RACE NM_004530.1 matrix metalloproteinase 2 824 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase) mir-143 3′RACE NM_015208.1 KIAA0874 protein 825 mir-143 3′RACE NM_002582.1 poly(A)-specific ribonuclease 826 (deadenylation nuclease) mir-143 3′RACE NM_000297.2 polycystic kidney disease 2 827 (autosomal dominant) mir-143 3′RACE NM_001175 Rho GDP dissociation inhibitor (GDI) 828 beta mir-143 3′RACE XM_166529 glucocorticoid induced transcript 1, 837 GLCCI1 mir-143 3′RACE- NM_001753.2 caveolin 1, caveolae protein, 22 kDa 791 2nt mir-143 3′RACE- NM_006732 FBJ murine osteosarcoma viral 803 2nt oncogene homolog B mir-143 3′RACE- NM_000100.1 cystatin B (stefin B) 807 2nt mir-143 3′RACE- NM_015959.1 CGI-31 protein 808 2nt mir-143 3′RACE- NM_004156.1 protein phosphatase 2 (formerly 2A), 814 2nt catalytic subunit, beta isoform mir-143 3′RACE- NM_031157 heterogeneous nuclear 815 2nt ribonucleoprotein A1 mir-143 3′RACE- NM_002582.1 poly(A)-specific ribonuclease 826 2nt (deadenylation nuclease) mir-143 3′RACE- NM_000297.2 polycystic kidney disease 2 827 2nt (autosomal dominant) mir-143 3′RACE- NM_006325.2 RAN, member RAS oncogene family 829 2nt mir-143 3′RACE- NM_004627.1 tryptophan rich basic protein 830 2nt mir-143 3′RACE- NM_012210.1 tripartite motif-containing 32 831 2nt mir-143 3′RACE- AJ131244.1 SEC24 related gene family, member A 832 2nt (S. cerevisiae) mir-143 3′RACE- NM_031267.1 cell division cycle 2-like 5 833 2nt (cholinesterase-related cell division controller) mir-143 3′RACE- AL049367.1 guanine nucleotide binding protein 835 2nt (G protein), gamma 12 mir-143 3′RACE- NM_001344 defender against cell death 1 836 2nt mir-131 3′RACE AK001214.1 hypothetical protein FLJ10352 1735 mir-131 3′RACE NM_001614 actin, gamma 1 (ACTG1), mRNA 1736 mir-131 3′RACE NM_001948.1 dUTP pyrophosphatase (DUT), mRNA 1737 mir-131 3′RACE NM_002387.1 mutated in colorectal cancers (MCC), 1738 mRNA mir-131 3′RACE NM_004109.1 ferredoxin 1 (FDX1), nuclear gene 1739 encoding mitochondrial protein, mRNA mir-131 3′RACE NM_004342.4 caldesmon 1 (CALD1), transcript 1740 variant 2, mRNA mir-131 3′RACE NM_005572.2 lamin A/C (LMNA), transcript variant 1741 2, mRNA mir-131 3′RACE NM_015640.1 PAI-1 mRNA-binding protein (PAI- 1742 RBP1), mRNA mir-131 3′RACE NM_017789.1 semaphorin 4C (SEMA4C), mRNA 1743 mir-131 3′RACE NM_144697.1 hypothetical protein BC017397 1744 (LOC148523), mRNA mir-131 3′RACE NM_173710 NADH dehydrogenase 3 (MTND3), mRNA 1745 mir-15a 5′RACE AF220018.1 Homo sapiens tripartite motif 1746 protein (TRIM2) mRNA mir-15a 5′RACE M98399.1 Human antigen CD36 mRNA 1747 mir-15a 5′RACE Y00281.1 Human mRNA for ribophorin I 1748

Because these RNA transcripts in Table 27 were identified as being bound by one of the mir-143, mir-131, or mir-15a miRNAs, these miRNAs are predicted to serve a regulatory role in expression or activity of these transcripts identified by RACE-PCR. Additional candidate human RNA targets can be identified in the same manner.

Example 21 Effects of Oligomeric Compounds on Adipocyte Differentiation Hallmark Genes in Differentiated Adipocytes

The effect of the oligomeric compounds of the present invention targeting miRNAs on the expression of markers of cellular differentiation was examined in differentiated adipocytes.

The effects of the oligomeric compounds of the present invention on the hallmark genes known to be upregulated during adipocyte differentiation assayed in Example 13 were also assayed in differentiated adipocytes. As previously described, the HSL, aP2, Glut4, and PPARγ genes play important rolls in the uptake of glucose and the metabolism and utilization of fats. Also as previously described, an increase in triglyceride content is another well-established marker for adipocyte differentiation. Human white preadipocytes (Zen-Bio Inc., Research Triangle Park, N.C.) were grown in preadipocyte media (ZenBio Inc.). After the cells reached confluence (approximately three days), they were exposed to differentiation media (Zen-Bio, Inc.) containing a PPAR-γ agonist, IBMX, dexamethasone, and insulin for three days. Cells were then fed Adipocyte Medium (Zen-Bio, Inc.), which was replaced at 2 to 3 day intervals. One day before transfection, 96-well plates were seeded with 3000 cells/well. Cells were then transfected on day nine post-differentiation, according to standard published procedures with 250 nM oligonucleotide in LIPOFECTIN™ (Invitrogen Corporation, Carlsbad, Calif.) (Monia et al., J. Biol. Chem. 1993 268(19):14514-22). Oligomeric compounds were tested in triplicate on each 96-well plate, and the effect of TNF-α, known to inhibit adipocyte differentiation, was also measured in triplicate. Oligomeric compound treatments and transfectant-only negative controls may be measured up to six times per plate. On day twelve post-differentiation, cells were washed and lysed at room temperature, and the expression of the four hallmark genes, HSL, aP2, Glut4, and PPARγ, as well as triglyceride (TG) accumulation were measured in adipocytes transfected with the uniform 2′-MOE phosphorothioate (PS) previously described in Example 13 as well as the chimeric gapmer oligomeric compounds targeting the mir-143 miRNA and the mir-143 pri-miRNA described in Example 16. On day twelve post-differentiation, cells were lysed in a guanadinium-containing buffer and total RNA was harvested. The amount of total RNA in each sample was determined using a Ribogreen Assay (Molecular Probes, Eugene, Oreg.). Real-time PCR was performed on the total RNA using primer/probe sets for the adipocyte differentiation hallmark genes Glut4, HSL, aP2, and PPARγ. Triglyceride levels as well as mRNA levels for each of the four adipocyte differentiation hallmark genes are expressed relative to control levels (control=treatment with ISIS-29848 (SEQ ID NO: 737)). The results of this experiment are shown in Table 28.

TABLE 28 Effects of oligomeric compounds targeting miRNAs on expression of adipocyte differentiation markers ISIS SEQ ID Number NO TG aP2 HSL Glut4 PPAR gamma 327876 294 1.16 0.67 0.81 3.53 1.28 327878 296 1.08 0.13 0.19 0.17 0.85 327880 298 1.12 1.14 0.93 0.76 1.86 327888 306 1.13 0.73 0.84 0.56 1.69 327889 307 1.09 1.12 0.77 0.99 1.63 327890 308 1.13 0.35 0.42 0.37 1.05 327892 310 1.23 0.81 0.62 0.42 1.01 327901 319 1.12 1.28 1.47 2.20 1.34 327903 321 1.12 0.56 0.53 0.36 0.91 327905 323 1.18 0.85 0.65 0.58 1.31 327913 331 1.12 1.05 1.09 1.52 1.29 327919 337 1.15 1.20 0.83 1.82 1.80 327922 340 1.48 0.91 1.01 0.61 0.99 327925 343 1.33 0.78 1.20 0.74 1.30 327933 351 1.63 1.58 1.30 2.12 1.60 327934 352 1.43 1.50 1.97 1.52 1.54 327939 357 1.33 1.16 1.08 0.72 1.89 327941 359 1.33 0.90 1.17 0.90 1.66 327954 372 1.46 1.23 1.35 0.61 1.46 328382 491 1.33 0.92 0.53 0.75 0.97 338664 491 1.72 0.77 1.01 1.08 1.06 340927 319 1.61 0.71 0.64 0.96 1.21

From these data, it was observed that the compound targeting the mir-203 miRNA (ISIS Number 327878), exhibited a sustained reduction in the hallmark marker genes at the 12^(th) day post differentiation. Treatment with this compound resulted in decreased expression of the aP2, HSL, Glut4 and PPARγ marker genes, indicating that this oligomeric compound may lead to reduced levels of mobilization of fatty acids from adipose tissue, and has the potential to ameliorate some of the symptoms of type 2 diabetes, obesity, hypertension, atherosclerosis, cardiovascular disease, insulin resistance, and certain cancers. Notably, the effect of treatment of differentiated adipocytes with this oligomeric compound targeting the mir-203 miRNA mirrors the effect of treating cells with the TNF-α positive control that inhibits adipocyte differentiation. This evidence suggests that the oligomeric compound targeting the mir-203 miRNA can act as a TNF-α mimetic compound, and potentially may be used in the suppression of cellular differentiation and the maintenance of cells in a quiescent state.

The oligomeric compound targeting the mir-203 miRNA was also tested in the insulin assay (see Example 18) and was observed to reduce expression of PEPCK-c, indicating that it may also be useful as an insulin mimetic and/or antidiabetic drug.

As an extension of these conclusions, one having ordinary skill in the art would appreciate that further modified oligomeric compounds could be designed to also target the mir-203 mature miRNA, or the pri-miRNA and pre-miRNA precursors. Such compounds are noted to be within the scope of the present invention.

Example 22 Effects of Oligomeric Compounds on Lymphocytic Leukemia Cells

Mir15-a-1 and mir-16-3 have been recently shown to reside in human chromosomal region (13q14) that is deleted in about 50% of chronic lymphocytic leukemia (CLL) patients. Mir-15 and 16 were found to be down-regulated in about 68% of CLL cases (Calin et al., Proc. Natl. Acad. Sci. USA, 2002, 99, 15524-15529, which is incorporated herein by reference in its entirety). CLL B-cells develop chemotherapy resistance over time, possibly due to a defective apoptosis pathway.

Using the 5′RACE method (described in Example 20), the CD36 mRNA was identified as one target regulated by mir-15 and/or mir-16 miRNAs. CD36 is a scavenger receptor involved in fat uptake by macrophages and adipocytes. CD36 is reported to be upregulated in some CLL cell lines, and its expression may correlate with tumor invasiveness.

If the apoptosis pathway is defective and the deletion or down-regulation of mir-15 and/or mir-16 play a role in CLL chemo-resistance, then addition of mir-15 and/or mir-16 should be able to induce apoptosis in CLL and increase drug-induced apoptosis. RNA oligonucleotide molecules ISIS Number 338963 (TAGCAGCACATAATGGTTTGTG; SEQ ID NO: 269) representing mir-15a-1/mir-15a-2, ISIS Number 338961 (TAGCAGCACATCATGGTTTACA; SEQ ID NO: 246) representing mir-15b, and ISIS Number 338965 (TAGCAGCACGTAAATATTGGCG; SEQ ID NO: 196) representing mir-16-1/mir-16-2/mir-16-3 were synthesized and deprotected. Additionally, RNA oligonucleotides bearing imperfect complementarity to these miRNA mimics (mimicking the imperfect complementarity found in the pri-miRNA) were also synthesized and deprotected. These imperfect complements were ISIS Number 338964 (TGCAGGCCATATTGTGCTGCCT; SEQ ID NO: 840), which is partially complementary to ISIS Number 338963 and represents the imperfect complement of mir-15a-1/mir-15a-2; ISIS Number 338962 (TGCGAATCATTATTTGCTGCTC; SEQ ID NO: 841), which is partially complementary to ISIS Number 338961 and represents the imperfect complement of mir-15b; ISIS Number 338966 (CTCCAGTATTAACTGTGCTGCTG; SEQ ID NO: 842), which is partially complementary to ISIS Number 338965 and represents the imperfect complement of mir-16-1 and mir-16-2; and ISIS Number 338967 (CACCAATATTACTGTGCTGCTT; SEQ ID NO: 843), which is partially complementary to ISIS Number 338965 and represents the imperfect complement of mir-16-3. These RNA molecules were diluted in water, and the concentration determined by A₂₆₀. Equimolar amounts of each of the miRNAs and their imperfect complementary RNA sequences were mixed together in the presence of Dharmacon 5× Universal buffer to form four “natural” double-stranded miRNA mimics. ISIS Number 338965 (SEQ ID NO: 196) was used twice; once, it was hybridized to ISIS Number 338966, and once it was hybridized to ISIS Number 338967, to form two different “natural” double-stranded miRNA mimics, Mir-16-1/Mir-16-2 and Mir-16-3, with imperfect complementarity. The mixture of four “natural” miRNA mimics was incubated for 1-5 minutes at 90° C. (the time depends on the volume of the mixture) and then incubated at 37° C. for one hour. A₂₆₀ readings were taken on the mixture for final concentration determination.

Heparinized peripheral blood from CLL patients was separated on a Ficoll density gradient to obtain greater than 95% pure CLL B-cells. These cells are tested for expression of the CD5/CD 19/CD23 antigens. Positive expression of these three antigens indicates that the cells are CLL B-cells (Pederson et al., Blood, 2002, 100, 2965, which is incorporated herein by reference in its entirety). Additionally, cytogenetic analysis can be performed to ascertain that the cells have the 13q deletion. A mixture of all four “natural” miRNA mimics at 2 μM each was electroporated into the cells. The cells were cultured in the presence or absence of apoptosis-inducing agents fludarabine A, or Dexamethasone (which are known to employ the intrinsic mitochondrial apoptotic pathway) or the antitumor agent CDDO-Im (reported to function through an alternative extrinsic apoptotic pathway) for 24 hours. Following incubation, apoptosis was monitored by annexin/PI double staining as outlined in FIG. 1 of Pederson et al., Blood, 2002, 100, 2965. The double-stranded RNA oligomeric compounds representing mir-15 and mir-16 miRNA mimics were observed to play a role in the induction of spontaneous as well as drug-induced apoptosis. Thus, oligomeric compounds of the present invention may be useful in the treatment of CD36-related diseases and conditions such as chronic lymphocytic leukemia and other cancers.

Example 23 Effect of Oligomeric Compounds Targeting miRNAs In Vivo

As described herein, leptin-deficient (ob/ob) mice, leptin receptor-deficient (db/db) mice and diet-induced obesity (DIO) mice are used to model obesity and diabetes. In accordance with the present invention, oligomeric compounds targeting mir-143, mir-131 (also known as mir-9) and mir-203 were tested in the ob/ob and db/db models. The ob/ob mice were fed a high fat diet and were subcutaneously injected with the oligomeric compounds of the invention or a control compound at a dose of 25 mg/kg two times per week for 6 weeks. Saline-injected animals, leptin wildtype littermates (i.e. lean littermates) and ob/ob mice fed a standard rodent diet served as controls. The physiological effects resulting from inhibition of target RNA, such as the effects of target inhibition on glucose and insulin metabolism and the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism, were assessed by methods disclosed herein. In brief, plasma levels of liver transaminases, cholesterol, triglycerides, free fatty acids and glucose were assessed weekly by tail bleed, with the tail bleed on week three taken under fasting conditions. After the treatment period, mice were sacrificed and liver, spleen, pancreas, muscle, kidney and heart, as well as brown adipose tissue (BAT) and white adipose tissue (WAT) tissues were collected. mRNA expression levels of the Glut4, aP2, HSL and PPARγ marker genes were evaluated. RNA isolation and target RNA expression level quantitation are performed as described.

Two oligomeric compounds targeting the mir-143 miRNA were compared for their effects on the physiological indications of obesity and diabetes. The oligomeric compound, ISIS Number 327901 (SEQ ID NO: 319), 22-nucleotides in length, targets the mature mir-143, and is a uniform 2′-MOE oligonucleotide with phosphorothioate internucleoside linkages throughout. The oligomeric compound ISIS Number 340927 (SEQ ID NO: 319) is a 5-10-7 gapmer also designed to target the mature mir-143 miRNA. The effects of these oligomeric compounds targeting mir-143 on several physiological parameters and markers of obesity and/or diabetes were examined in vivo. Potential effects on food consumption were also monitored.

Plasma cholesterol levels were observed to slightly decrease over time in ob/ob mice treated with the gapmer oligomeric compound ISIS Number 340927 (SEQ ID NO: 319) targeted to mir-143. Similarly, plasma triglyceride and plasma glucose levels were generally slightly lower in ob/ob mice treated with this compound as compared to untreated mice, or mice treated with control compounds. mRNA expression levels of the Glut4, aP2 and HSL marker genes were slightly reduced by both oligomeric compounds ISIS Number 327901 and ISIS Number 340927 targeting mir-143. Thus, these oligomeric compounds targeting mir-143 may be useful compounds in the treatment of obesity or diabetes.

In addition, Northern blot analyses were performed to quantitate the expression of mature mir-143 in kidney samples of ob/ob mice treated with oligomeric compounds of the present invention. The mir-143 specific DNA oligonucleotide probe (SEQ ID NO: 319) described above was used to detect expression levels of the mir-143 miRNA in ob/ob mice treated (twice weekly at 25 mg/kg) with ISIS Numbers 327901, the uniform 2′-MOE oligomeric compound, or ISIS Number 340927, the 5-10-7 gapmer compound, both targeted to mir-143, versus saline treated animals or animals treated with ISIS 342672 (SEQ ID NO: 789), a uniform 2′-MOE scrambled negative control oligomeric compound. Expression levels were normalized against the U6 RNA and the expression levels of saline treated animals were set at 100%. Most notably, in kidney samples from ob/ob mice treated with ISIS Number 327901, the uniform 2′-MOE oligomeric compound targeted to mir-143 exhibited a nearly 40% decrease in in vivo expression levels of the mature mir-143 miRNA. In kidney samples from mice treated with the gapmer oligomeric compound targeting mir-143, ISIS Number 340927, a 23% reduction in in vivo expression levels of the mature mir-143 miRNA was observed.

Oligomeric compounds targeting the mir-131/mir-9 and the mir-203 miRNAs were also tested for their effects on the physiological indicators or markers of obesity and diabetes. The oligomeric compound, ISIS Number 327892 (SEQ ID NO: 310), targeted to mir-131/mir-9,21-nucleotides in length, is a uniform 2′-MOE oligonucleotide with phosphorothioate internucleoside linkages throughout. The oligomeric compound ISIS Number 340926 (SEQ ID NO: 310) is a 5-10-6 gapmer oligomeric compound also designed to target the mir-131/mir-9 miRNA. The oligomeric compound ISIS Number 327878 (SEQ ID NO: 296) targeted to mir-203, 22-nucleotides in length, is a uniform 2′-MOE oligonucleotide with phosphorothioate internucleoside linkages throughout. The oligomeric compound ISIS Number 345349 (SEQ ID NO: 296) is a 5-10-7 gapmer oligomeric compound also designed to target the mir-203 miRNA. The effects of these oligomeric compounds were examined in vivo in the ob/ob model. Potential effects on food consumption were also monitored.

Fed plasma glucose levels in ob/ob mice treated with the oligomeric compounds ISIS Number 327892 (SEQ ID NO: 310) and ISIS Number 340926 (SEQ ID NO: 310) targeted to mir-131/mir-9, and ISIS Number 327878 (SEQ ID NO: 296) and ISIS Number 345349 (SEQ ID NO: 296) targeted to mir-203 were observed to be reduced beginning at approximately four weeks after the start of treatment and continuing to decrease on week five as compared to untreated mice, or mice treated with control compounds. Triglyceride levels were also observed to be reduced over time in mice treated with ISIS 340926 and 345349, the gapmer oligomeric compounds targeted to mir-131/mir-9 and mir-203, respectively. No signs of liver toxicity were indicated by weekly measurements of plasma transaminases upon treatment of ob/ob mice with any of the oligomeric compounds targeting mir-143, mir-203 or mir-131/mir9.

ob/ob mice in the fasted state on day 19 after treatment with the oligomeric compounds ISIS Number 327892 (SEQ ID NO: 310) and ISIS Number 340926 (SEQ ID NO: 310) targeted to mir-131/mir-9 also exhibited significant reductions in plasma glucose levels. Notably, the gapmer oligomeric compound ISIS Number 340926 (SEQ ID NO: 310) targeted to mir-131/mir-9 was even more potent than the corresponding uniform 2′-MOE oligonucleotide ISIS Number 327892 (SEQ ID NO: 310).

Furthermore, a decrease in food consumption was observed by the third week and this reduced level was maintained in the fourth week post-treatment of ob/ob mice with these oligomeric compounds. Therefore, the oligomeric compounds targeting the mir-131/mir-9 and mir-203 miRNAs have potential use as appetite suppressants, as well as in the treatment of obesity or diabetes.

The oligomeric compounds ISIS Number 327901 and ISIS Number 340927 both targeting mir-143, ISIS Number 327892 and ISIS Number 340926 both targeting mir-131/mir-9, and ISIS Number 327878 and ISIS Number 345349 both targeting mir-203 were also tested in db/db mice. Although treatment of db/db mice with the gapmer compounds targeting mir-143, mir-203 or mir-131/mir9 resulted in an approximately 2-fold increase in liver transaminases in db/db mice, the uniform 2′-MOE oligomeric compounds targeting mir-143, mir-203 or mir-131/mir-9 were not found to cause liver toxicity in db/db mice, as assessed by weekly measurements of plasma transaminase levels.

Additional oligomeric compounds targeting miRNAs were studied in ob/ob mice. Six week old ob/ob mice were treated (dose=25 mg/kg, twice weekly for four weeks) with uniform 2′-MOE and gapmer oligomeric compounds targeting mir-143, mir-23b, mir-221, let-7a, and mir-29b, and compared to saline treated animals or animals treated with ISIS 342672 (SEQ ID NO: 789), a uniform 2′-MOE scrambled negative control oligomeric compound bearing 13 base mismatches to mir-143. Expression levels were normalized against the U6 RNA and the expression levels of saline treated animals were set at 100%. Fed plasma samples were taken bi-weekly by tail bleed, and plasma levels of liver transaminases, cholesterol, triglycerides, free fatty acids and glucose were assessed, with the tail bleed on week three taken under fasting conditions. Ob/ob mice were treated with ISIS Numbers 327901 and 340927, the uniform 2′-MOE and gapmer oligomeric compounds, respectively, targeting mir-143 are described above. Additionally, ob/ob mice were also treated with the following compounds: ISIS Number 327889 (SEQ ID NO: 307), a phosphorothioate uniform 2′-MOE oligomeric compound, and ISIS Number 340925 (SEQ ID NO: 307), a 2′-MOE 5-10-8 gapmer oligomeric compound, each targeting mir-23b; ISIS Number 327919 (SEQ ID NO: 337), a uniform 2′-MOE oligomeric compound, and ISIS Number 345384 (SEQ ID NO: 337), a phosphorothioate 2′-MOE 5-10-8 gapmer oligomeric compound, each targeting mir-221; ISIS Number 327903 (SEQ ID NO: 321), a uniform 2′-MOE oligomeric compound, and ISIS Number 345370 (SEQ ID NO: 321), a phosphorothioate 2′-MOE 5-10-7 gapmer oligomeric compound, each targeting let-7a; and ISIS Number 327876 (SEQ ID NO: 294), a uniform 2′-MOE oligomeric compound, and ISIS Number 345347 (SEQ ID NO: 294), a phosphorothioate 2′-MOE 5-10-8 gapmer oligomeric compound, each targeted to mir-29b-1.

Ob/ob mice treated with the gapmer compounds ISIS 340925 and ISIS 345384, targeting mir-23b and mir-221, respectively, exhibited reductions in plasma glucose levels in the fed state at weeks two and four, as compared to untreated mice, or mice treated with control compounds. Furthermore, mice treated with ISIS 340925 exhibited a decrease in triglycerides in the fourth week. Ob/ob mice treated with ISIS 340925 did not exhibit an increase in plasma transaminases at weeks two or four. Thus, the oligomeric compounds ISIS Numbers 340925 and 345384 may be useful as agents for the treatment of obesity and/or diabetes.

In addition, Northern blot analyses were performed to quantitate the expression of mir-23b in kidney samples of ob/ob mice treated with oligomeric compounds of the present invention. To detect the mir-23b target, a target-specific DNA oligonucleotide probe with the sequence GTGGTAATCCCTGGCAATGTGAT (SEQ ID NO: 307) was synthesized by IDT (Coralville, Iowa). The oligo probes were 5′ end-labeled with T4 polynucleotide kinase with (γ-³²P) ATP (Promega). The mir-23b specific DNA oligonucleotide probe was used to detect expression levels of the mir-23b miRNA in ob/ob mice treated (twice weekly at 25 mg/kg) with ISIS Numbers 327889, the uniform 2′-MOE oligomeric compound, or ISIS Number 340925, the 5-10-8 gapmer compound, both targeted to mir-23b, versus saline treated animals or animals treated with a control oligomeric compound, ISIS Number 116847 (CTGCTAGCCTCTGGATTTGA; SEQ ID NO: 844), a uniform 5-10-5 2′-MOE gapmer targeting an unrelated gene, PTEN. Expression levels were normalized against the U6 RNA and the expression levels of saline treated animals were set at 100%. Most notably, in kidney samples from ob/ob mice treated with ISIS Number 327889, the uniform 2′-MOE oligomeric compound targeted to mir-23b exhibited a nearly 64% decrease in in vivo expression levels of the mir-23b miRNA. In kidney samples from mice treated with the gapmer oligomeric compound targeting mir-23b, ISIS Number 340925, a 41% reduction in in vivo expression levels of the mir-23b miRNA was observed.

As described, supra, the C57BL/6 mouse strain is reported to be susceptible to hyperlipidemia-induced atherosclerotic plaque formation, and when these mice are fed a high-fat diet, they develop diet-induced obesity (DIO). Accordingly, the DIO mouse model is useful for the investigation of obesity and development of agents designed to treat these conditions. In one embodiment of the present invention, oligomeric compounds targeting miRNAs were tested in the DIO model. Normal C57/BL6 male mice were fed a high fat diet (40% fat, 41% carbohydrate, 18% protein) for 12 weeks before the study began. DIO mice were then randomized by weight and insulin values. Initial body fat composition was determined by Dual X-ray Absorptiometry (DEXA) Scan. DIO mice were then subcutaneously injected with oligomeric compounds of the invention at a dose of 25 mg/kg, twice weekly. DIO mice were treated with oligomeric compounds ISIS Numbers 327901 and 340927 targeting mir-143, ISIS Numbers 327892 and 340926 targeting mir-131/mir-9, ISIS Numbers 327878 and ISIS Number 345349 targeting mir-203, and ISIS Numbers 327889 and 340925, targeting mir-23b. As negative controls, “scrambled” oligomeric compounds were also designed: ISIS Number 342672 contains 13 mismatches with respect to the mature mir-143 miRNA; ISIS Number 353607 (ACTAGTTTTTCTTACGTCTGA; herein incorporated as SEQ ID NO: 845) is a phosphorothioate 5-10-6 2′-MOE gapmer oligomeric compound containing 12 mismatches with respect to mir-131/mir-9; ISIS Number 353608 (CTAGACATTAGCTTTGACATCC; herein incorporated as SEQ ID NO: 846) is a phosphorothioate 5-10-7 2′-MOE gapmer oligomeric compound containing 16 mismatches with respect to mir-203. DEXA scans were also performed at weeks 0, 3 and 5 after treatment with the oligomeric compounds to assess the fat mass to lean mass ratio. The effects of target inhibition on levels of plasma glucose and insulin, liver transaminases, cholesterol and triglycerides, were also assessed weekly by tail bleed, and after the treatment period, mice were sacrificed and liver and kidney heart, as well as white adipose tissue (WAT) tissues collected. The mRNA expression levels of the Glut4, aP2, HSL and PPARγ marker genes are also assessed. Treatment of DIO mice with the uniform 2′-MOE oligomeric compounds ISIS 327901 targeting mir-143, ISIS 327892 targeting mir-131/mir9, ISIS 327878 targeting mir-203, and ISIS 327889 targeting mir-23b did not appear to cause liver toxicity in these mice as assessed by weekly measurements of plasma transaminase levels. Similarly, the gapmer oligomeric compounds ISIS 340927 targeting mir-143, and ISIS 340926 targeting mir-131/mir-9, 340925 did not cause significant increases in liver toxicity, and the gapmer compound ISIS 340925 targeting mir-23b caused only an approximately 2-fold increase in the liver transaminase AST. Interestingly, the gapmer compounds ISIS Numbers 340927 targeting mir-143, 340926 targeting mir-131/mir-9, 345349 targeting mir-203, and 340925, targeting mir-23b were all effective at reducing insulin levels at the two and four week time points, as compared to saline-treated control mice. Furthermore, some improvement in body composition (a reduction in body weight and fat mass) was observed. These data from the DIO model suggest that oligomeric compounds targeting mir-143, mir-131/mir-9, mir-203 and mir-23b may be useful as agents for the treatment of obesity and/or diabetes.

Having the information disclosed herein, one of ordinary skill in the art would comprehend that of other classes of inhibitors targeting mir-143, mir-209, mir-131/mir-9 and mir-23b miRNAs, such as antibodies, small molecules, and inhibitory peptides, can be assessed for their effects on the physiological indicators of diseases in in vivo models, and these inhibitors can be developed for the treatment, amelioration or improvement of physiological conditions associated with a particular disease state or condition. Such inhibitors are envisioned as within the scope of the instant invention.

Example 24 Effects of Oligomeric Compounds on Cell Cycling

Cell Cycle Assay:

Cell cycle regulation is the basis for various cancer therapeutics. Cell cycle checkpoints are responsible for surveillance of proper completion of certain steps in cell division such as chromosome replication, spindle microtubule attachment and chromosome segregation, and it is believed that checkpoint functions are compromised in some cancerous cells. Furthermore, because the shift from quiescence to an actively growing state as well as the passage through mitotic checkpoints are essential transitions in cancer cells, most current chemotherapy agents target dividing cells. For example, by blocking the synthesis of new DNA required for cell division, an anticancer drug can block cells in S-phase of the cell cycle. These chemotherapy agents impact many healthy organs as well as tumors. In some cases, a cell cycle regulator will cause apoptosis in cancer cells, but allow normal cells to undergo growth arrest and therefore remain unaffected. Loss of tumor suppressors such as p53 sensitizes cells to certain anticancer drugs; however, cancer cells often escape apoptosis. Further disruption of cell cycle checkpoints in cancer cells can increase sensitivity to chemotherapy while allowing normal cells to take refuge in G1 and remain unaffected. A goal of these assays is to determine the effects of oligomeric compounds on the distribution of cells in various phases of the cell cycle.

In some embodiments, the effects of several oligomeric compounds of the present invention were examined in the normal human foreskin fibroblast BJ cell line, the mouse melanoma cell line B16-F10 (also known as B16 cells), as well as the breast carcinoma cell line, T47D. These cell lines can be obtained from the American Type Culture Collection (Manassas, Va.). BJ cells were routinely cultured in MEM high glucose with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate and supplemented with 10% fetal bovine serum, 0.1 mM non-essential amino acids and 1.0 mM sodium pyruvate (all media and supplements from Invitrogen Life Technologies, Carlsbad, Calif.). B16-F10 cells were routinely cultured in DMEM high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). T47D cells were cultured in DMEM High glucose media (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum. Cells were routinely passaged by trypsinization and dilution when they reached 80 to 90% confluence. Cells were plated on collagen-coated 24-well plates (Falcon-Primaria #3047, BD Biosciences, Bedford, Mass.) at approximately 50,000 cells per well and allowed to attach to wells overnight.

As a negative control, a random-mer oligomeric compound, 20 nucleotides in length, ISIS 29848 (SEQ ID NO: 737) was used. In addition, a positive control, ISIS 183891 (CCGAGCTCTCTTATCAACAG; herein incorporated as SEQ ID NO: 847) was included; ISIS 183891 targets kinesin-like 1 (also known as Eg5) and inhibits cell cycle progression. Eg5 is known to induce apoptosis when inhibited. ISIS 29248 and ISIS 183891 are chimeric oligomeric compounds (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings” (a “5-10-5 gapmer”). The wings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the compound. All cytidine residues are 5-methylcytidines. ISIS 340348 (CTACCTGCACGAACAGCACTTT; herein incorporated as SEQ ID NO: 848) is a uniform 2′-MOE phosphorothioate oligomeric compound targeting the mir-93 miRNA, and ISIS 340365 (TACTTTATATAGAACACAAG; herein incorporated as SEQ ID NO: 849) is a 5-10-5 gapmer phosphorothioate oligomeric compound targeting the mir-92-2 miRNA.

Oligomeric compounds were mixed with LIPOFECTIN™ (Invitrogen Life Technologies, Carlsbad, Calif.) in OPTI-MEM™ (Invitrogen Life Technologies, Carlsbad, Calif.) to achieve a final concentration of 150 nM of oligomeric compound and 4.5 μg/ml LIPOFECTIN™. Before adding to cells, the oligomeric compound, LIPOFECTIN™ and OPTI-MEM™ were mixed thoroughly and incubated for 0.5 hrs. The medium was removed from the plates and each well was washed in 250 μl of phosphate-buffered saline. The wash buffer in each well was replaced with 250 μL of the oligomeric compound/OPTI-MEM™/LIPOFECTIN cocktail. Control cells received LIPOFECTIN™ only. The plates were incubated for 4 hours at 37° C., after which the medium was removed. 100 μl of full growth medium was added to each well. After 72 hours, routine procedures were used to prepare cells for flow cytometry analysis and cells were fixed with ethanol and stained with propidium iodide to generate a cell cycle profile using a flow cytometer. The cell cycle profile was analyzed with the ModFit program (Verity Software House, Inc., Topsham Me.).

Fragmentation of nuclear DNA is a hallmark of apoptosis and produces an increase in cells with a hypodiploid DNA content. Cells with a hypodiploid DNA content are categorized as “subG1.” The cells in the G1, G2/M and S phases are considered to be cycling, and cells in the subG1 and aneuploid categories are considered to have left the cell cycle. An increase in cells in G1 phase is indicative of a cell cycle arrest prior to entry into S phase; an increase in cells in S phase is indicative of cell cycle arrest during DNA synthesis; and an increase in cells in the G2/M phase is indicative of cell cycle arrest just prior to or during mitosis. Data are shown in Table 29 and expressed as percentage of cells in each phase of the cell cycle.

TABLE 29 Effects of oligomeric compounds targeting miRNAs on cell cycling SEQ Pri- an- ISIS # ID # miRNA SubG1 G1 S G2/M euploid UTC N/A N/A 8.1 59.6 27.5 12.9 7.3 ISIS-29848 737 N/A 9.6 57.8 26.5 15.6 12 n-mer ISIS-183891 847 Kinesin- 20.8 33.1 39.2 27.6 11.5 Positive like 1/Eg5 control 327878 296 mir-203 17.3 39.1 40.8 20 11.9 327888 306 mir-108-1 13.3 53.7 29.5 16.7 12.9 327889 307 mir-23b 8.2 53.1 32.5 14.4 10.5 327901 319 mir-143 12 34.7 44.9 20.3 13.6 327902 320 mir-192-1 10.6 50.7 33.9 15.3 13.4 327903 321 let-7a-3 11 53.7 30.9 15.4 13.4 327904 322 mir-181a 8.6 54.4 29.5 16.2 15.6 327905 323 mir-205 8.5 56.9 28.1 15 14.7 327906 324 mir-103-1 15.2 46.1 33 20.9 15.8 327907 325 mir-26a 17.8 49.5 32.8 17.6 17.8 327908 326 mir-33a 5.6 55.4 29.2 15.3 13.1 327909 327 mir-196-2 7.9 52.6 30.1 17.3 16.3 327910 328 mir-107 9.3 49.5 33 17.5 13.1 327911 329 mir-106 10.9 49.9 30.1 20 16.5 327914 332 mir-130a 8.5 55.8 28.9 15.3 16.2 327919 337 mir-221 10.8 54.3 30.3 15.4 16 327922 340 mir-19b-2 10 50.4 30.7 18.9 16.8 327928 346 mir-29a-1 6.6 56 27.9 16 15.9 327933 351 mir-145 10.2 49.6 31.3 19.1 15.9 327934 352 mir-213 6.6 54.4 28.2 17.4 17 327941 359 mir-181b 8.2 57.2 29.9 12.9 15.8 327951 369 mir-15a-1 4.3 60.9 24.8 14.3 16.7 328342 451 mir-203 4.8 62.3 24.9 12.8 15.2 328362 471 mir-108-1 9.1 51.2 33.6 15.1 12.9 328364 473 mir-23b 1.9 61.5 24.2 14.3 15.1 328382 491 mir-143 2.9 59.8 25.7 14.4 14.8 328388 497 let-7a-3 4.0 57.5 28 14.6 14.5 328394 503 mir-181a 2.4 59.5 24.5 16 18.3 328396 505 mir-205 4.6 56.8 28.2 15 19.8 328419 528 mir-221 6.0 51.2 32.5 16.3 17.9 328423 532 mir-19b-2 4.9 52.9 32.4 14.8 15.3 328424 533 mir-19b-2 3.1 61.9 23.7 14.4 16.9 328436 545 mir-29a-1 3.5 59.2 26.9 13.9 17.4 328644 553 mir-145 7.2 58.4 27.6 14 17.5 328691 600 mir-145 7.7 60.5 24.4 15.1 16.6 328697 606 mir-181b 2.4 57.6 26.4 16 13.5 328773 682 mir-15a-2 2.7 56.4 26.9 16.7 11.7 340348 848 mir-93 14.1 53.9 31.8 14.3 12.3 340365 849 mir-92-2 4.3 55.2 29.4 15.4 18.3

From these data, it is evident that treatment with the oligomeric compounds targeting mir-143, ISIS Number 327901 (SEQ ID NO: 319); mir-203, ISIS Number 327878 (SEQ ID NO: 296); mir-103-1, ISIS Number 327906 (SEQ ID NO: 324); mir-106, ISIS Number 327911 (SEQ ID NO: 329); and mir-145, ISIS Number 327933 (SEQ ID NO: 351) resulted in an increased percentage of cells in the G2/M phase, indicating that these oligomeric compounds arrest or delay the cell cycle at or just prior to mitosis, potentially activating a mitotic checkpoint.

Treatment with the oligomeric compounds targeting mir-26a, ISIS Number 327907 (SEQ ID NO: 325); mir-205, ISIS Number 328396 (SEQ ID NO: 505); mir-181a, ISIS Number 328394 (SEQ ID NO: 503); and mir-92-2, ISIS Number 340365 (SEQ ID NO: 849) resulted in higher than average percentages of aneuploid cells, indicating that these oligomeric compounds interfere with proper chromosome segregation.

Treatment with the oligomeric compounds targeting mir-203, ISIS Number 327878 (SEQ ID NO: 296); mir-103-1, ISIS Number 327906 (SEQ ID NO: 324); mir-26a, ISIS Number 327907 (SEQ ID NO: 325); and mir-93, ISIS Number 340348 (SEQ ID NO: 848) resulted in an increased percentage of cells with hypodiploid DNA content (SubG1 phase) indicating that the oligomeric compound treatment may induce apoptotic events.

The effects of several oligomeric compounds of the present invention were also examined in the HeLa and A549 human carcinoma cell lines, both of which can be obtained from the American Type Culture Collection (Manassas, Va.).

In some embodiments, HeLa cells were plated on collagen-coated 24-well plates at 50,000-60,000 cells per well, and allowed to attach to wells overnight. In some embodiments, HeLa cells were synchronized by double thymidine block (cells were washed three times with PBS, then grown in 10% FBS containing 2 mM thymidine; then 19 hours later, cells were washed three times in PBS, 10% FBS for 9 hours; cells were then incubated in 10% FBS, 2 mM thymidine for 15 hours; then washed three times with PBS, 10% FBS and samples were taken every two hours over a 16 hour period). A portion of each time sample was fixed with ethanol and treated with propidium iodide and subjected to FACs analysis for determination of the percentage of cells in each phase of the cell cycle. Distinctive peaks were observed for G0-, S-, Early G2/M-, Late G2/M-, and G1-phases of the cell cycle at 0-, 4-, 6-, 8-, and 12-hours, respectively, indicating that the cells were synchronized. HeLa cells treated with 10 μM cisplatin or 100 ng/ml nocodazole were used as controls for G1-phase and late G2/M-phases, respectively. From the remaining portion of each of these time samples, total RNA was isolated and used to assess the expression of cell cycle marker mRNAs using the real-time RT-PCR methods and/or used to screen microarrays to assess the expression of miRNAs over the course of the cell cycle. It was observed that several miRNAs are expressed in a cell-cycle-dependent manner. Shown in Table 30 are the mRNA levels of the E2F1 transcription factor and topoisomerase 2A (Top2A), which vary over the course of the cell cycle and can be used for comparison to the experimental groups for the confirmation of cell cycle phase. Data are an average of three trials.

TABLE 30 Expression levels of cell cycle markers treatment E2F1 mRNA Top2A mRNA 10 uM cisplatin 102 15 100 ng/ml nocodazole 23 176 0 hrs (G0-phase) 100 100 4 hrs (S-phase) 81 105 6 hrs (early G2/M-phase) 39 221 8 hrs (late G2/M-phase) 50 254 12 hrs (G1-phase) 61 124

In some embodiments, HeLa cells were also treated with oligomeric compounds targeting miRNAs. As described above, oligomeric compounds were mixed with LIPOFECTIN™ in OPTI-MEM™ (Invitrogen Life Technologies, Carlsbad, Calif.) to a final concentration of 150 nM of oligomeric compound and 6 μg/ml LIPOFECTIN™. Before adding to cells, the oligomeric compound, LIPOFECTIN™ and OPTI-MEM™ were mixed thoroughly and incubated for 0.5 hrs. The medium was removed from the plates. Each well was washed in 250 μl of PBS. The wash buffer in each well was replaced with 250 μL of the oligomeric compound/OPTI-MEM™/LIPOFECTIN cocktail. Control cells received LIPOFECTIN™ only. The plates were incubated for 4 hours at 37° C., after which the medium was removed. 1000 μl of full growth medium was added to each well. After 24 hours (Table 31) or 48 hours (Table 32), cells were prepared for flow cytometry analysis to generate a cell cycle profile. The cell cycle profile was analyzed with the ModFit program (Verity Software House, Inc., Topsham Me.).

The random-mer ISIS 29848 (SEQ ID NO: 737) was used as a negative control, and ISIS 183891 (SEQ ID NO: 847), targeting kinesin-like 1/Eg5, was included as a positive control. Results of these experiments are shown in Tables 31 and 32. Data are expressed as percentage of cells in each phase relative to the untreated control (UTC); values above 100 are considered to indicate a delay or arrest in that phase of the cell cycle. Table 31 shows the results from cells sampled 24 hours after oligomeric compound treatment, and Table 32 shows the results from cells sampled 48 hours after oligomeric compound treatment. In some cases, the same oligomeric compound was tested in repeated experiments.

TABLE 31 Effects of oligomeric compounds targeting miRNAs on cell cycling (24 hours) SEQ % cells in cell cycle phase Pri-miRNA ISIS # ID # subG1 G1 S G2/M aneuploid UTC N/A N/A 100 100 100 100 100 n-mer  29848 737 120 116 81 108 76 Kinesin-like 1/Eg5 183891 847 251 21 109 231 95 collagen, type I, 338797 624 197 101 79 148 193 alpha 1/ hypothetical miRNA-144 hypothetical miRNA-039 338666 493 235 123 63 158 102 hypothetical miRNA-111 328111 413 62 127 75 99 50 hypothetical miRNA-111 338750 577 107 148 76 97 166 hypothetical miRNA-142 328115 417 177 90 87 147 59 hypothetical miRNA-154 328119 421 75 100 94 112 83 hypothetical miRNA-154 328724 633 155 91 90 135 197 hypothetical miRNA-179 328749 658 312 126 82 110 138 hypothetical miRNA-179 328780 689 124 96 87 136 149 hypothetical miRNA-181 328136 438 330 125 81 88 51 hypothetical miRNA-181 338833 660 232 150 56 142 185 let-7a-3 327903 321 118 92 104 106 98 let-7a-3 328388 375 120 110 83 115 85 mir-100-1 327957 497 197 91 88 145 66 mir-100-1 328707 616 188 36 93 195 166 mir-103-1 327906 324 228 153 47 107 65 mir-103-1 328397 506 134 93 86 142 91 mir-106 327911 329 158 130 62 122 104 mir-106 328403 512 284 70 85 197 53 mir-106 328403 512 189 86 75 179 82 mir-107 327910 328 174 154 42 118 73 mir-108-1 328362 471 114 101 87 126 66 mir-10a 327949 367 194 82 84 172 68 MiR-125a, Mouse 341787 852 221 113 75 144 165 mir-127, Mouse 341788 853 303 154 54 140 114 mir-130b 328687 596 231 80 98 131 149 mir-130b 338769 596 188 171 61 103 133 mir-131-2/mir-9 327892 310 153 86 111 103 80 mir-131-2/mir-9 328369 310 84 100 88 125 71 mir-131-2/mir-9 340926 478 286 98 91 121 83 mir-133b 338713 540 93 152 72 101 187 mir-141 338741 568 157 141 73 112 166 mir-143 327901 319 108 101 94 110 90 mir-143 328382 491 81 118 76 116 78 mir-143 328382 491 226 102 80 144 202 mir-143 340927 319 118 121 75 111 88 mir-143 340927 319 131 128 71 106 87 mir-145 327933 351 192 102 83 131 92 mir-145 327933 351 190 90 91 140 47 mir-145 328644 553 71 113 84 109 68 mir-145 345395 351 247 54 82 222 77 mir-149, Mouse 341785 854 125 152 92 53 158 mir-152 328727 636 245 133 81 105 161 mir-152 338809 636 106 159 82 69 210 mir-16-3 327877 295 154 107 66 159 62 mir-17/mir-91 327885 303 151 129 63 121 55 mir-181a-1 327904 322 114 99 102 99 89 mir-182 328744 653 229 31 108 167 111 mir-182 338826 653 145 148 79 90 138 mir-192-1 327902 320 178 57 106 176 66 mir-192-1 327902 320 175 44 121 163 98 mir-192-1 328383 492 314 55 82 222 92 mir-192-1 328383 492 289 63 97 183 98 mir-192-1 338665 340 173 85 76 175 193 mir-19b-2 327922 492 131 97 96 114 104 mir-19b-2 328424 533 60 110 85 112 74 mir-203 327878 296 124 96 94 122 73 mir-203 328342 451 192 33 95 238 67 mir-205 327905 323 144 99 88 129 50 mir-205 327905 323 149 94 95 121 98 mir-205 328396 505 97 94 87 139 88 mir-205 338678 505 162 122 75 131 202 mir-211 327946 364 225 90 84 156 43 mir-211 328674 583 564 125 93 84 69 mir-211 338756 583 137 147 75 99 166 mir-213/mir-181a-2 327934 352 278 87 85 160 55 mir-213/mir-181a-2 327934 352 204 118 66 137 77 mir-213/mir-181a-2 328647 556 140 101 92 119 140 mir-216 327956 374 120 124 68 120 61 mir-216 328759 668 239 88 78 168 184 mir-22 327896 314 121 83 103 128 65 mir-22 328374 483 198 54 115 162 97 mir-220 327944 362 165 85 110 111 50 mir-221 327919 337 85 92 103 109 96 mir-221 328419 528 87 109 79 124 77 mir-23a 338836 663 153 185 53 105 150 mir-23b 327889 307 122 104 102 87 82 mir-23b 340925 307 151 103 89 117 73 mir-26a-1 327907 325 224 119 77 111 75 mir-26a-1 345373 325 196 66 94 176 68 mir-29b-1 327876 294 103 98 104 95 66 mir-29b-1 327876 294 149 93 92 131 75 mir-29b-1 328337 446 107 106 88 113 104 mir-29b-1 328337 446 99 108 88 109 64 mir-29b-2 328339 448 235 77 102 143 61 mir-29c 338690 517 149 124 78 123 194 mir-30a 328084 585 381 43 104 163 101 mir-30b 328676 585 139 99 86 134 169 mir-30b 338758 743 113 129 81 108 190 mir-30d 328421 530 288 47 105 200 70 mir-33a 327908 326 138 98 99 106 114 mir-92-1 327897 315 143 114 80 115 69 mir-92-1 327897 315 180 128 74 100 54 mir-92-2 340365 849 109 125 71 114 84 mir-95 (Mourelatos) 340350 855 218 183 54 104 94 mir-96 338637 464 88 170 70 84 188

TABLE 32 Effects of oligomeric compounds targeting miRNAs on cell cycling (48 hours) % cells in cell cycle phase Pri-miRNA ISIS # SEQ ID # subG1 G1 S G2/M aneuploid UTC N/A N/A 100 100 100 100 100 n-mer  29848 737 86 87 121 117 109 Kinesin-like 1/Eg5 183891 847 173 19 124 331 72 collagen, type I, 338797 624 813 66 124 168 175 alpha 1/ hypothetical miRNA-144 hypothetical miRNA-039 338666 493 1832 44 136 217 125 hypothetical miRNA-111 328111 413 371 84 126 119 90 hypothetical miRNA-111 338750 577 201 99 101 103 190 hypothetical miRNA-142 328115 417 195 92 114 107 86 hypothetical miRNA-154 328119 421 767 75 145 124 81 hypothetical miRNA-154 328724 633 653 70 134 140 155 hypothetical miRNA-179 328749 658 962 37 129 246 65 hypothetical miRNA-179 328780 689 917 83 130 110 133 hypothetical miRNA-181 328136 438 140 83 133 113 85 hypothetical miRNA-181 338833 660 1091 44 106 258 154 let-7a-3 327903 321 74 102 95 98 94 let-7a-3 328388 375 112 99 101 102 126 mir-100-1 327957 497 864 65 169 127 85 mir-100-1 328707 616 1486 46 134 213 155 mir-103-1 327906 324 57 100 98 103 83 mir-103-1 328397 506 74 97 101 109 96 mir-106 327911 329 65 99 96 109 101 mir-106 328403 512 863 61 177 131 85 mir-106 328403 512 108 82 148 106 80 mir-107 327910 328 53 99 91 111 92 mir-108-1 328362 471 87 96 104 108 97 mir-10a 327949 367 773 66 157 138 71 MiR-125a, Mouse 341787 852 707 55 126 197 153 mir-127, Mouse 341788 853 748 76 105 163 116 mir-130b 328687 596 1119 55 174 131 171 mir-130b 338769 596 482 76 116 149 194 mir-131-2/mir-9 327892 310 121 74 150 129 79 mir-131-2/mir-9 328369 310 72 99 95 109 109 mir-131-2/mir-9 340926 478 68 83 120 131 106 mir-133b 338713 540 426 95 104 109 194 mir-141 338741 568 185 100 101 99 170 mir-143 327901 319 93 98 104 103 104 mir-143 328382 491 71 102 92 103 109 mir-143 328382 491 350 83 122 120 133 mir-143 340927 319 95 91 107 121 113 mir-143 340927 319 83 91 107 122 108 mir-145 327933 351 91 76 135 138 86 mir-145 327933 351 438 80 133 123 75 mir-145 328644 553 52 101 101 98 82 mir-145 345395 351 213 51 192 157 87 mir-149, Mouse 341785 854 1148 82 126 116 166 mir-152 328727 636 846 68 152 124 140 mir-152 338809 636 345 86 110 129 157 mir-16-3 327877 295 755 59 152 168 80 mir-17/mir-91 327885 303 456 78 129 133 76 mir-181a-1 327904 322 116 87 126 114 80 mir-182 328744 653 1774 31 78 334 171 mir-182 338826 653 696 61 124 182 137 mir-192-1 327902 320 1176 39 171 208 81 mir-192-1 327902 320 202 44 166 205 87 mir-192-1 328383 492 303 53 217 124 90 mir-192-1 328383 492 940 54 178 150 90 mir-192-1 338665 340 1629 40 89 292 149 mir-19b-2 327922 492 81 96 105 109 91 mir-19b-2 328424 533 89 103 91 101 111 mir-203 327878 296 50 89 119 114 92 mir-203 328342 451 189 55 115 225 107 mir-205 327905 323 719 48 194 150 67 mir-205 327905 323 100 78 143 122 99 mir-205 328396 505 88 89 114 119 129 mir-205 338678 505 1158 81 78 188 179 mir-211 327946 364 431 72 150 129 76 mir-211 328674 583 1663 69 160 109 134 mir-211 338756 583 311 90 121 100 169 mir-213/mir-181a-2 327934 352 752 62 156 152 92 mir-213/mir-181a-2 327934 352 155 66 148 155 117 mir-213/mir-181a-2 328647 556 589 69 153 118 136 mir-216 327956 374 184 91 106 121 110 mir-216 328759 668 1744 50 31 343 148 mir-22 327896 314 886 55 140 194 66 mir-22 328374 483 787 65 157 143 71 mir-220 327944 362 490 75 129 144 78 mir-221 327919 337 104 80 122 139 104 mir-221 328419 528 83 99 96 107 112 mir-23a 338836 663 811 52 152 169 165 mir-23b 327889 307 133 78 137 130 101 mir-23b 340925 307 89 87 130 109 93 mir-26a-1 327907 325 116 92 111 115 94 mir-26a-1 345373 325 116 75 132 145 119 mir-29b-1 327876 294 41 87 120 119 100 mir-29b-1 327876 294 251 76 141 126 69 mir-29b-1 328337 446 66 92 105 119 108 mir-29b-1 328337 446 662 73 143 135 74 mir-29b-2 328339 448 678 73 153 123 92 mir-29c 338690 517 413 91 110 112 190 mir-30a 328084 585 1028 20 168 241 57 mir-30b 328676 585 366 86 118 118 172 mir-30b 338758 743 267 103 99 92 153 mir-30d 328421 530 1103 30 202 198 64 mir-33a 327908 326 61 99 98 105 93 mir-92-1 327897 315 134 100 103 95 84 mir-92-1 327897 315 125 94 114 105 63 mir-92-2 340365 849 71 99 94 109 129 mir-95 (Mourelatos) 340350 855 1144 76 126 134 125 mir-96 338637 464 239 90 109 117 210

Several oligomeric compounds were observed to result in an arrest or delay of the cell cycle, in some cases correlating with a cell-cycle-dependent expression profile as determined by miRNA microarray analysis.

For example, from these data, it was observed that treatment of HeLa cells with oligomeric compounds (MOE-gapmers and fully modified MOEs) targeting miRNAs caused an increase in the percentage of cells exhibiting a subG1-phase or aneuploid DNA content, indicating aberrant chromosome segregation. Treatment with oligomeric compounds ISIS Number 338797 (SEQ ID NO: 624) targeted to hypothetical miRNA-144, ISIS Number 338833 (SEQ ID NO: 660) targeted to hypothetical miRNA-181, and ISIS Number 328759 (SEQ ID NO: 668) targeted to mir-216, each appeared to cause an induce chromosome missegregation events at both the 24-hour and 48-hour timepoints. Thus, these compounds may be useful in triggering a checkpoint arrest in rapidly dividing cells, potentially useful in the treatment of hyperproliferative disorders such as cancer.

It was also observed that other oligomeric compounds (MOE-gapmers and fully modified MOEs) targeting miRNAs appeared to induce an arrest or delay in the G1-, S-, or G2/M-phases of the cell cycle. By miRNA microarray analysis, expression levels of the mir-205 miRNA were observed to increase in the S- and G1-phases of the cell cycle in HeLa cells. Treatment of HeLa cells with the oligomeric compound ISIS Number 327905 (SEQ ID NO: 323), targeting the mir-205 miRNA, was observed to arrest or delay the cell cycle in S-phase at the 48-hour time point, suggesting that the mir-205 miRNA may play a role in regulating DNA replication. It was also observed that treatment of HeLa cells with the oligomeric compound ISIS Number 338678 (SEQ ID NO: 505), targeted to the mir-205 pri-miRNA, resulted in an arrest or delay primarily in the G2/M-phase of the cell cycle, suggesting that this oligomeric compound may interfere with processing of the miRNA precursor into a mature miRNA, which appears to have an impact on mitosis.

Treatment of HeLa cells with oligomeric compounds ISIS Number 327892 (SEQ ID NO: 310), targeting mir-131/mir-9, and ISIS Number 327934 (SEQ ID NO: 352), targeting mir-213/mir-181a-2, was observed to arrest or delay the cell cycle in G2/M- and S-phases at the 48-hour time point, suggesting that the mir-131/mir-9 and mir-213/mir-181a-2 miRNAs are involved in regulating DNA replication and entry into mitosis.

Treatment of HeLa cells with oligomeric compound ISIS Number 345373 (SEQ ID NO: 325), targeting miR-26a-1, was observed to arrest or delay cells mainly in the G2/M-phase at 24 hrs after oligonucleotide-treatment, and at 48 hrs after oligonucleotide-treatment to arrest or delay cells mainly in S-phase of the cell cycle, suggesting that miR-26a-1 is involved in mitosis and that cells making it through a first round of cell division may harbor errors that cause them to arrest during a new round of DNA replication.

By miRNA microarray analysis, expression levels of the mir-145 miRNA were observed to increase in the G2/M-phase of the cell cycle in HeLa cells, and treatment of HeLa cells with the oligomeric compounds ISIS Number 327933 (SEQ ID NO: 351), a uniform 2′-MOE compound, and ISIS Number 345395 (SEQ ID NO: 351), a chimeric 2′-MOE gapmer compound, both targeting the mir-145 miRNA, were observed to arrest or delay the cell cycle in G2/M-phase at the 24-hour time point and at subG1-phase at the 48-hour time point, suggesting that the mir-145 miRNA plays a role in mitosis and that cells making it through a first round of cell division may harbor errors that cause them to arrest before a new round of DNA replication.

By miRNA microarray analysis, expression levels of the mir-192-1 miRNA were observed to increase in the G2/M-phase of the cell cycle in HeLa cells, and treatment of HeLa cells with the oligomeric compounds ISIS Number 327902 (SEQ ID NO: 320), a uniform 2′-MOE compound, and ISIS Number 328383 (SEQ ID NO: 492), a chimeric 2′-MOE gapmer compound, targeted against the mir-192-1 miRNA and the mir-192-1 precursor, respectively, were observed to arrest or delay the cell cycle in the G2/M-phase at 24-hours after oligonucleotide treatment, and at both the S- and G2/M-phases at the 48-hour time point, suggesting that the mir-192 miRNA is involved in mitosis, and that cells making it through a first round of cell division may harbor errors that cause them to arrest during a new round of DNA replication. A uniform 2′-MOE oligomeric compound ISIS Number 338665 targeting the mir-192-1 precursor was also observed to induce a G2/M-phase arrest at both time points.

Treatment of HeLa cells with the oligomeric compound ISIS Number 328744 (SEQ ID NO: 653), targeting the mir-182 miRNA, was observed to arrest or delay the cell cycle in G2/M-phase at 48-hours after oligonucleotide treatment, suggesting that the mir-182 miRNA plays a role in regulating mitosis.

Treatment of HeLa cells with the oligomeric compound ISIS Number 328421 (SEQ ID NO: 530), targeting miR-30d was also observed to arrest or delay cells mainly in the G2/M-phase at the 24-hour time point and at both the S- and G2/M-phases at the 48-hour time point after oligonucleotide treatment, suggesting that the mir-30d miRNA is involved in mitosis, and that a cell division error arising from the first round of division may allow cells to pass through mitosis and initiate a second round of division, but then a cell cycle checkpoint is set off before the cells are able to complete DNA synthesis.

Treatment of HeLa cells with the oligomeric compound ISIS Number 328403 (SEQ ID NO: 512), targeting mir-106 was also observed to arrest or delay cells in the G2/M-phase at the 24-hour time point and at both the S- and G2/M-phases at the 48-hour time point after oligonucleotide treatment, suggesting that the mir-106 miRNA is involved in mitosis, and that a cell division error arising from the first round of division may allow cells to pass through mitosis and initiate a second round of division, but then a cell cycle checkpoint is set off before the cells are able to complete DNA synthesis. Interestingly, the cell cycle regulatory transcription factor E2F1 mRNA is reported to be a target of the mir-106 miRNA (Lewis et al., Cell, 2003, 115, 787-798).

Treatment of HeLa cells with the oligomeric compound ISIS Number 328759 (SEQ ID NO: 668), targeting the mir-216 miRNA, was observed to arrest or delay the cell cycle in G2/M-phase at both 24- and 48-hours after oligonucleotide treatment, suggesting that the mir-216 miRNA plays a role in regulating mitosis.

Treatment of HeLa cells with the oligomeric compound ISIS Number 328342 (SEQ ID NO: 451), targeting the mir-203 miRNA, was observed to arrest or delay the cell cycle in G2/M-phase at both 24- and 48-hours after oligonucleotide treatment, suggesting that the mir-203 miRNA plays a role in regulating mitosis.

Treatment of HeLa cells with the oligomeric compound ISIS Number 328707 (SEQ ID NO: 616), targeting miR-100-1 was also observed to arrest or delay cells mainly in the G2/M-phase at both 24- and 48-hours after oligonucleotide treatment, suggesting that the miR-100-1 miRNA plays a role in regulating mitosis.

Dose Responsiveness:

In accordance with the present invention, certain oligomeric compounds targeting miRNAs were selected for dose response studies. Using the cell cycle assay described above, the cell cycle profiles of HeLa or A549 cells treated with varying concentrations of oligomeric compounds of the present invention were assessed.

HeLa cells were treated with 25-, 50-, 100- or 150 nM of the oligomeric compounds ISIS Numbers 327902 (SEQ ID NO: 320) and 328383 (SEQ ID NO: 492), both targeted against mir-192, and ISIS 327905 (SEQ ID NO: 323), targeting mir-205, and ISIS 328403 (SEQ ID NO: 512), targeting mir-106. Cells treated with increasing concentrations of oligomeric compounds were compared to untreated cells, to assess the dose-dependency of the observed delay or arrest. The random-mer ISIS 29848 was used as a negative control. Cells were prepared for flow cytometry 48-hours after oligonucleotide treatment, as described, supra. Oligomeric compounds targeted to miRNAs were tested in quadruplicate, and ISIS 29848 was tested in triplicate; data is presented as an average of the replicates. Results of these dose response studies are shown in Table 33, where data are expressed as percentage of cells in each phase.

TABLE 33 Dose response of oligomeric compounds targeting miRNAs on cell cycling (48 hours) Dose oligomeric % cells in cell cycle phase ISIS # compound SubG1 G1 S G2/M Aneuploid Untreated 25 nM 1.3 56 24 20 12 control 50 nM 1.4 56 24 20 14 (UTC) 100 nM 1.6 57 24 19 11 150 nM 1.6 57 23 20 15  29848 25 nM 2.0 55 25 20 12 50 nM 1.5 56 25 19 12 100 nM 3.2 52 28 20 13 150 nM 4.2 48 31 21 15 327902 25 nM 1.6 57 23 19 13 50 nM 2.4 51 30 20 14 100 nM 3.1 43 30 27 11 150 nM 6.3 36 36 28 12 327905 25 nM 1.7 57 24 18 12 50 nM 2.1 50 30 20 12 100 nM 2.5 46 30 24 14 150 nM 4.5 38 38 24 12 328383 25 nM 1.9 57 25 18 12 50 nM 1.3 56 25 18 13 100 nM 9.3 36 34 30 10 150 nM 11.8 29 36 34 11 328403 25 nM 1.5 58 24 18 13 50 nM 1.1 53 27 20 14 100 nM 3.5 48 29 23 11 150 nM 8.2 37 40 24 13

From these data, it was observed that 48-hours after treatment of HeLa cells with increasing doses of each of these four oligomeric compounds targeting miRNAs, a dose-responsive delay or arrest resulted, exhibited as an increasing percentage of cells in the S- and G2/M-phases of the cell cycle. Concomitant decreases in the percentage of cells in G1-phase of the cell cycle and increases in the percentage of hypodiploid (subG1) cells were also observed. Likewise, a dose-responsive G2/M delay or arrest was observed in A549 cells treated with 25-, 50-, 100-, or 150 nM of the oligomeric compounds ISIS 327902, ISIS 328383 and ISIS Number 328342.

In a further study, A549 cells were treated with 25-, 50-, 100- or 150 nM of the oligomeric compounds ISIS Numbers 338637 (SEQ ID NO: 464) targeted against mir-96, and ISIS 338769 (SEQ ID NO: 596) targeted against mir-130b, ISIS 338836 (SEQ ID NO: 663) targeted against mir-23a, and ISIS 340350 (SEQ ID NO: 855) targeted against mir-95 (Mourelatos). Cells treated with increasing concentrations of oligomeric compounds were compared to untreated cells, to assess the dose-responsiveness of the observed delay or arrest. The random-mer ISIS 29848 was used as a negative control. Cells were prepared for flow cytometry 24-hours after oligonucleotide treatment. Results of these dose response studies are shown in Table 34, where data are expressed as percentage of cells in each phase relative to the untreated control cells in that phase.

TABLE 34 Dose response of oligomeric compounds targeting miRNAs on cell cycling (24 hours) Dose oligomeric % cells in cell cycle phase ISIS # compound SubG1 G1 S G2/M Aneuploid 29848 25 nM 90 121 86 87 76 50 nM 91 116 88 93 90 100 nM 272 125 74 112 116 150 nM 507 126 71 119 84 338637 25 nM 89 100 99 101 99 50 nM 86 110 89 107 120 100 nM 67 126 73 115 146 150 nM 216 123 66 144 135 338769 25 nM 62 101 94 114 101 50 nM 82 114 81 122 132 100 nM 130 124 75 113 157 150 nM 341 117 71 145 184 338836 25 nM 76 97 103 97 99 50 nM 232 113 89 98 111 100 nM 68 117 80 116 153 150 nM 178 117 69 149 114 340350 25 nM 91 102 100 95 120 50 nM 158 128 67 126 80 100 nM 267 125 60 155 107 150 nM 402 128 40 211 108

From these data, it was observed that 24-hours after treatment of A549 cells with increasing doses of the oligomeric compounds ISIS Numbers 338637 (SEQ ID NO: 464) targeted against mir-96, and ISIS 338769 (SEQ ID NO: 596) targeted against mir-130b, ISIS 338836 (SEQ ID NO: 663) targeted against mir-23a, and ISIS 340350 (SEQ ID NO:855) targeted against mir-95 (Mourelatos), a dose-responsive delay or arrest resulted, exhibited as an increasing percentage of cells in the G2/M-phases of the cell cycle. Concomitant decreases in the percentage of cells in S-phase of the cell cycle and increases in the percentage of hypodiploid (subG1) cells were also observed.

In further studies, additional cell lines were treated with oligomeric compounds targeted against miRNAs to assess the effects of each oligomeric compound on cell cycling. BJ, B16, T47D, and HeLa cells were cultured and transfected as described above. T47D cells are deficient in p53. T47Dp53 cells are T47D cells that have been transfected with and selected for maintenance of a plasmid that expresses a wildtype copy of the p53 gene (for example, pCMV-p53; Clontech, Palo Alto, Calif.), using standard laboratory procedures. BJ cells were treated with 200 nM of each oligomeric compound, and T47D, T47Dp53, HeLa, and B16 cells were treated with 150 nM of each oligomeric compound. The human foreskin fibroblast BJ cell line represents a non-cancer cell line, while HeLa, T47D, T47Dp53 cells and the mouse melanoma cell line B16-F10 represent cancerous cell lines. For comparison, oligomeric compounds ISIS 183891 (SEQ ID NO: 847) and ISIS 285717 (TCGGTTCTTTCCAAGGCTGA; herein incorporated as SEQ ID NO: 857), both targeting the kinesin-like 1/Eg5 mRNA, involved in cell cycling, were used as positive controls. The random-mer ISIS 29848 was used as a negative control. Additionally, the oligomeric compounds ISIS Number 25690 (ATCCCTTTCTTCCGCATGTG; herein incorporated as SEQ ID NO: 858) and ISIS Number 25691 (GCCAAGGCGTGACATGATAT; herein incorporated as SEQ ID NO: 859), targeted to nucleotides 3051-3070 and 3085-3104, respectively, of the mRNA encoding the Drosha RNase III (GenBank Accession NM_(—)013235.2, incorporated herein as SEQ ID NO: 860) were also tested. ISIS Number 25690 and ISIS Number 25691 are 5-10-5 2′-MOE gapmer compounds, 20 nucleotides in length, with phosphorothioate internucleoside linkages throughout the oligomeric compound. All cytidine residues are 5-methylcytidines. Transfections were performed using the methods described herein. Cells were prepared for flow cytometry 48-hours after oligonucleotide treatment. Results of these studies are shown in Table 35, where data are expressed as percentage of cells in each phase relative to the untreated control cells in that phase.

TABLE 35 Effects of oligomeric compounds targeting miRNAs on cell cycling (48 hours) SEQ % cells in cell cycle phase Cell ID Sub line ISIS # NO target G1 G1 S G2/M aneuploid BJ 29848 737 N/A 187 100 99 100 105 B16 29848 737 N/A 143 98 98 110 99 HeLa 29848 737 N/A 403 83 113 106 155 T47D 29848 737 N/A 86 95 113 98 155 T47Dp53 29848 737 N/A 173 121 75 97 93 BJ 183891 847 kinesin-like 1/eg5 422 58 173 287 158 B16 285717 857 kinesin-like 1/eg5 627 72 78 220 178 HeLa 183891 847 kinesin-like 1/eg5 1237 22 95 211 161 T47D 183891 847 kinesin-like 1/eg5 85 55 84 156 161 T47Dp53 183891 847 kinesin-like 1/eg5 351 71 53 189 84 HeLa 25690 858 Drosha, RNAse III 64 119 89 87 140 T47D 25690 858 Drosha, RNAse III 97 97 80 113 140 T47Dp53 25690 858 Drosha, RNAse III 193 97 108 114 144 BJ 25691 859 Drosha, RNAse III 183 94 116 125 209 B16 25691 859 Drosha, RNAse III 316 116 83 99 105 HeLa 25691 859 Drosha, RNAse III 881 53 141 113 203 T47D 25691 859 Drosha, RNAse III 125 94 104 104 203 T47Dp53 25691 859 Drosha, RNAse III 212 130 66 93 95 HeLa 338797 624 hypothetical 144 104 89 115 125 miRNA-144 HeLa 338666 493 hypothetical miRNA 214 92 98 130 151 039 HeLa 338833 660 hypothetical miRNA 255 87 100 136 136 181 HeLa 328707 616 mir-100-1 125 103 87 122 140 BJ 328403 512 mir-106 81 102 95 92 114 B16 328403 512 mir-106 112 111 88 99 92 HeLa 328403 512 mir-106 89 125 89 80 175 T47D 328403 512 mir-106 49 104 112 89 175 T47Dp53 328403 512 mir-106 140 114 87 94 89 HeLa 341787 852 MiR-125a, Mouse 324 88 96 145 177 T47D 328687 596 mir-130b 142 101 92 115 169 T47Dp53 338769 596 mir-130b 116 103 87 123 87 B16 327933 351 mir-145 104 109 84 116 130 BJ 345395 351 mir-145 132 100 97 104 115 B16 345395 351 mir-145 147 106 87 115 150 HeLa 345395 351 mir-145 87 108 96 95 139 BJ 328744 653 mir-182 125 94 111 127 158 B16 328744 653 mir-182 153 108 87 110 115 HeLa 328744 653 mir-182 1057 53 110 213 178 T47D 328744 653 mir-182 85 90 87 118 191 T47Dp53 328744 653 mir-182 90 130 59 101 100 BJ 327902 320 mir-192-1 91 99 88 108 82 B16 327902 320 mir-192-1 151 112 88 98 101 HeLa 327902 320 mir-192-1 94 108 96 93 162 T47D 327902 320 mir-192-1 102 75 120 116 162 T47Dp53 327902 320 mir-192-1 155 100 98 102 97 HeLa 338665 492 mir-192-1 322 92 92 142 138 HeLa 328342 451 mir-203 103 96 89 138 96 BJ 327905 323 mir-205 105 100 77 109 102 B16 327905 323 mir-205 142 107 89 106 94 HeLa 327905 323 mir-205 55 108 99 90 164 T47D 327905 323 mir-205 81 97 101 103 164 T47Dp53 327905 323 mir-205 109 112 80 104 103 HeLa 338678 505 mir-205 129 103 94 105 132 BJ 328759 668 mir-216 164 91 117 141 160 B16 328759 668 mir-216 132 104 91 110 126 HeLa 328759 668 mir-216 797 40 82 203 223 T47D 328759 668 mir-216 123 86 87 122 223 T47Dp53 328759 668 mir-216 423 99 93 108 109 HeLa 327896 314 mir-22 95 103 94 106 144 HeLa 338836 660 mir-23a 303 97 96 121 114 HeLa 328084 743 mir-30a 286 89 92 153 125 HeLa 340350 855 mir-95 132 101 94 112 177 (Mourelatos)

When treatment of cells with oligomeric compounds resulted in greater than 750% cells in subG1 phase, these oligomeric compounds were deemed to be “hits,” in that they appear to cause an increase in apoptosis, resulting in hypodiploid DNA contents. When treatment of cells with oligomeric compounds resulted in greater than 140% cells in G1-phase, these oligomeric compounds were deemed “hits,” as they appeared to cause an arrest or delay in G1-phase and/or blocked entry into S-phase of the cell cycle. When treatment of cells with oligomeric compounds resulted in greater than 140% cells in S-phase, these oligomeric compounds were deemed “hits,” as they appeared to cause an arrest or delay in DNA synthesis. When treatment of cells with oligomeric compounds resulted in greater than 140% cells in G2/M phase, these oligomeric compounds were deemed “hits,” as they appeared to cause an arrest or delay in the transition into mitosis, and/or in cell division, itself.

From these data, it was observed that 48-hours after treatment of the various cell lines with the oligomeric compounds, ISIS Number 183891 targeting the kinesin-like 1/Eg5 mRNA results in a delay or arrest in G2/M phase of the cell cycle for all cell lines. Treatment of HeLa cells with ISIS Number 25691, targeted against the Drosha RNase III mRNA, resulted in an increased percentage of cells in S-phase as well as a significant percentage of cells in the subG1 and aneuploid categories, indicating that this oligomeric compound may interfere with DNA replication and/or maintenance of the integrity of the proper complement of genetic material.

In HeLa cells, ISIS 341787 (SEQ ID NO: 852) targeted against mir-125a (mouse), resulted in an arrest or delay in G2/M as well as an increased percentage of cells in the subG1 and aneuploid categories, indicating that this oligomeric compound may interfere with cell division and equal chromosome segregation during mitosis.

In HeLa cells treated with ISIS 328744 (SEQ ID NO: 653) targeted against mir-182, an increase in the percentage of cells in the G2/M-phase of the cell cycle as well as in the subG1 category was observed, indicating that this oligomeric compound may interfere with cell division and equal chromosome segregation during mitosis. Notably, genetically normal cells (BJ and T47Dp53cells) were not affected by ISIS Number 328744, indicating that the oligomeric compound targeting miR-182 may selectively cause a cell cycle delay or arrest in cancer cells and not normal cells, and suggesting that this compound may be particularly useful as a therapeutic agent in the treatment of hyperproliferative disorders such as cancer.

In HeLa cells treated with ISIS 328759 (SEQ ID NO: 668) targeted against mir-216, a delay or arrest resulted in the G2/M-phase of the cell cycle was observed, as well as an increase in the percentage of cells in the subG1 and aneuploid categories, indicating that this oligomeric compound may interfere with cell division and equal chromosome segregation during mitosis.

Thus, it was observed that treatment of HeLa cells with oligomeric compounds targeting miRNAs is a effective means of identifying compounds that can block progression through various stages of the cell cycle. Notably, a transient increase in G1-phase was observed 24 hours after treatment of HeLa cells with oligomeric compounds targeting miRNAs; for example, oligomeric compounds ISIS Numbers 338769, 338836, 340350, and 338637 caused a transient increase in the percentage of cells delayed or arrested in G1-phase at the 24-hour time point, which, by the 48-hour time point, had shifted to a delay or arrest in S-phase. It was also noted that multiple oligomeric compounds targeting the same miRNA have the same effect on cell cycling. It was also observed that uniform 2′-MOE as well as 2′-MOE chimeric gapmer oligomeric compounds targeting the mature miRNA, as well as uniform 2′-MOE oligomeric compounds targeting the pri-miRNA often have the same effect.

Oligomeric compounds that delay, arrest or prevent cell cycle progression or induce apoptosis may be useful as therapeutic agents for the treatment of hyperproliferative disorders, such as cancer, cancer, as well as diseases associated with a hyperactivated immune response.

It is understood that BJ, B16, HeLa, A549, HMECs, T47D, T47Dp53, MCF7 or other cell lines can be treated with oligomeric compounds designed to mimic miRNAs in studies to examine their effects on progression through the cell cycle. Such oligomeric compounds are within the scope of the present invention.

Example 25 A Bioinformatic Approach to Identification of miRNA Targets

Several candidate RNA transcripts identified using the RACE-PCR methods described in Example 20 were the basis for a bioinformatic analysis of predicted targets bound to and/or potentially regulated by miRNAs. The complementarity between the miRNA used as a primer and the 3′-UTR of the RNA transcript identified by RACE-PCR was assessed using several methods. Transcripts identified by RACE-PCR were also analyzed using the FASTA sequence alignment program (accessible through the internet at, for example, www.ebi.ac.uk/fasta33) to find the best alignment between complementary sequences of the transcript and the miRNA used as a primer for RACE-PCR. When, using the default parameters, the FASTA alignment program resulted in the identification of the actual primer binding site (PBS) within the 3′-UTR of the RNA transcript as the sequence most complementary to the miRNA used as a primer in the RACE-PCR method, the candidate miRNA target transcript was specified by a plus sign (for example, see the “mir-143/PBS complementary?” column in Table 36 below). When the FASTA program failed to align the actual PBS with the sequence most complementary to the miRNA used in the RACE-PCR, the candidate miRNA target transcript was specified by a minus sign. When the FASTA program could be made to align with the sequence most complementary to the miRNA used in the RACE-PCR by decreasing the stringency of the FASTA program parameters, the candidate miRNA target transcript was specified by “±”.

A global alignment was also performed to assess whether the sequence of the PBS within the RNA transcript identified by RACE-PCR was conserved between human and mouse orthologs of the RNA target. For example, in Table 36, below, strong conservation of PBS in the human and murine orthologs (homology from 80-100%) was indicated by a plus sign; moderate conservation (homology between 70-80%) was indicated by “±”, and a minus sign indicates homology below 70%.

A variety of algorithms can be used to predict RNA secondary structures based on thermodynamic parameters and energy calculations. For example, secondary structure prediction can be performed using either M-fold or RNA Structure 2.52. M-fold can be accessed through the Internet at, for example, www.ibc.wustl.edu/-zuker/ma/form2.cgi or can be downloaded for local use on UNIX platforms. M-fold is also available as a part of GCG package. RNA Structure 2.52 is a windows adaptation of the M-fold algorithm and can be accessed through the Internet at, for example, 128.151.176.70/RNAstructure.html. The RNA Structure 2.52 program was used to analyze a series of 30-base fragments spanning the entire length of the human RNA transcript and their potential to hybridize with the miRNA used as a primer in the RACE-PCR, allowing the prediction of the lowest absolute free energy peak representing the most likely site of hybridization (including bulged regions) between the miRNA and the RNA target. If the free energy peak representing the hybridization between the miRNA and the PBS of the RNA transcript identified by RACE-PCR was among the top five peaks predicted by the RNA Structure 2.52 program, the transcript was given a plus sign, “+”. If the free energy peak representing the hybridization between the miRNA and the PBS was in the top five to ten peaks predicted by RNA Structure 2.52, the transcript was given a plus/minus sign, “±”, and if the peak representing the hybridization between the miRNA and the PBS was below the top ten peaks predicted by RNA Structure 2.52, the transcript was given a minus sign, “−”.

A list of the RNA transcript targets identified by RACE-PCR employing the mir-143 miRNA as a specific primer is shown in Table 36.

TABLE 36 Potential RNA targets of the mir-143 miRNA SEQ RNA RNA ID PBS Structure mir-143/PBS transcript target NO: conserved? peak? complementary? Matrix 819 + − + metalloproteinase 2 Sec24 829 − +/− + Tripartite motif- 828 +/− + +/− containing 32 RAN 824 +/− + + Cystatin B 802 − + + Glucocorticoid 839 + +/− + induced transcript 1 Protein 809 + + + phosphatase 2 Polycystic kidney 822 − − − disease 2 Mannose-6- 801 +/− − + phosphate receptor Mitotic control 817 + + − protein dis3 homolog Chromosome 14 813 + +/− − ORF 103 Rho GDP 823 − − − dissociation inhibitor beta Glyoxalase I 816 + + + Zinc finger protein 818 + +/− + 36, C3H type-like 1 LIM domain only 4 804 + + +

Note that four genes (Sec24, cystatin B, polycystic kidney disease 2, and Rho GDP dissociation inhibitor beta) did not have murine orthologs to compare in a global analysis of the PBS. Because these RNA transcripts were identified as being bound by the mir-143 miRNA used as a primer in the RACE-PCR approach previously described, the mir-143 miRNA is predicted to serve a regulatory role in expression or activity of one or more or all of these RNA transcripts. Of particular note are three targets, protein phosphatase 2, glyoxalase I, and LIM domain only 4 (LMO4) mRNAs, for which all three analyses yielded a positive result. That all three parameters assessed yielded a positive result suggests that these mRNAs are probable targets of mir-143.

The well-studied C. elegans lin-4 miRNA interaction with its lin-28 mRNA target was used as the starting point for a bioinformatics approach to the identification of miRNA binding sites in target nucleic acids. Lin-4 has been experimentally determined to bind at a single site on the lin-28 mRNA. Herein, as a primary determinant of miRNA-target interactions, it was hypothesized that the bimolecular hybridization free energies (ΔG°₃₇) of the interaction of the miRNA with a true target site would be more negative than the ΔG°₃₇ of other interactions of the miRNA with other binding sites. The nucleotide sequence of the lin-28 mRNA was assessed by computationally deriving 30-nucleotide windows, starting with the first nucleotide of the sequence and defining the first nucleotide in each window by shifting 1 nucleotide in the 3′ direction. Each window was assessed by hybridizing the 30-nucleotide sequence in the window with the lin-4 miRNA and disallowing unimolecular interactions, thereby spanning the entire length of the lin-28 mRNA, and the resulting ΔG°₃₇ value was plotted against the start position of the window. It was observed that the bimolecular hybridization between the true lin-4 binding site and the lin-28 mRNA had the lowest ΔG°₃₇ value, supporting our hypothesis and our bioinformatic approach to the identification of miRNA binding sites in target nucleic acids.

The mitogen-activated protein kinase 7/extracellular signal-regulated kinase 5 (ERK5) (GenBank Accession NM_(—)139032.1, incorporated herein as SEQ ID NO: 861) mRNA transcript was previously computationally predicted to be regulated by mir-143 miRNA binding in the 3′-UTR regions (Lewis et al., Cell, 2003, 115, 787-798). In order to identify mir-143 binding sites in the ERK5 mRNA, a bimolecular hybridization free energy assessment was performed by performing a hybridization walk to assess possible mir-143 binding sites along the entire length of the ERK5 mRNA. A strong negative ΔG°₃₇ value (−20.1) was found at the previously predicted mir-143 binding site in the 3′-UTR, lending further support to our method. Surprisingly, two additional, and novel, mir-143 binding sites with more negative ΔG°₃₇ values, as well as a third mir-143 binding site with a comparable ΔG°₃₇ value were also identified. Using the ERK5 sequence (GenBank Accession NM_(—)139032.1) as a reference, these binding sites encompass nucleotides 937-966 with a ΔG°₃₇ value of (−22.8), nucleotides 2041-2070 with a ΔG°₃₇ value of (−20.6) and nucleotides 2163-2192 with a ΔG°₃₇ value of (−19.3). See FIG. 1. Thus, three novel mir-143 binding sites (and, thus, a potential regulatory sites) were identified within the coding sequence of the ERK5 gene. Thus, this method of screening for miRNA binding sites by a bimolecular hybridization free energy assessment can be used to confirm previously predicted sites, and further allows the identification of novel miRNA target nucleic acid binding sites. It is believed that this method may more closely mimic the energetic mechanism by which a miRNA scans a target nucleic acid to find its interaction site. In subsequent experiments, the predicted mir-143 binding sites within the ERK5 coding sequence were also tested using the reporter system described below.

Example 26 Northern Analysis of miRNA Expression

As described in the adipocyte differentiation assay, the oligomeric compounds ISIS Number 327889 (SEQ ID NO: 307), targeted to mir-23b, and ISIS Number 327876 (SEQ ID NO: 294), targeted to mir-29b-1, were found to reduce the expression of several hallmark genes of adipocyte differentiation, indicating that mir-23b and mir-29b-1 may play a role in adipocyte differentiation, and that oligomeric compounds targeting these miRNAs may be useful as agents blocking cellular differentiation. Therefore, the expression of mir-23b and mir-29b was assessed by Northern blot of total RNA from multiple tissues. To detect the mir-23b and mir-29b-1 targets, target specific DNA oligonucleotide probes with the sequences GTGGTAATCCCTGGCAATGTGAT (SEQ ID NO: 307) and AACACTGATTTCAAA TGGTGCTA (SEQ ID NO: 294), respectively, were synthesized by IDT (Coralville, Iowa). The oligo probes were 5′ end-labeled with T4 polynucleotide kinase with (γ-³²P) ATP (Promega). To normalize for variations in loading and transfer efficiency membranes are stripped and probed for U6 RNA. Total RNA from mouse and human tissues as well as total RNA from human adipocytes and HepG2 cells was probed in Northern blot analyses, using methods described in Example 14.

By Northern analyses, the mir-23b miRNA was found to be most highly expressed in human kidney tissue as well as in adipose tissue from ob/ob mice, and was also highly expressed in human liver, adipocytes, preadipocytes and HepG2 cells. Moderate expression of mir-23b was also noted in murine kidney tissue. The mir-29b-1 miRNA was found to be most highly expressed in human and mouse kidney, and was also expressed in human liver, adipocytes, preadipocytes, and HepG2 cells, as well as in murine adipose tissue and liver. Levels of both the mir-23b and mir-29b-1 miRNAs were also found to be upregulated in human differentiated adipocytes.

Similarly, target specific DNA oligonucleotide probes for mir-16, mir-15a, and let-7a were designed and used in Northern blot analyses to assess expression of these miRNAs in human and mouse tissues. The mir-16 and mir-15a miRNAs were each found to be most highly expressed in human spleen, heart, testes, and kidney, and expression was also observed in liver as well as HEK293 and T47D cells. Additionally, low levels of expression of the mir-16 miRNA were observed in NT2 cells. The let-7a miRNA was most highly expressed in human and murine kidney, and expression was also observed in human and murine liver. Additionally, low levels of let-7a expression were found in HepG2 cells.

To detect the mir-21 miRNA in Northern blot analyses, a target specific DNA oligonucleotide probe with the sequences TCAACATCAGTCTGATAAGCTA (SEQ ID NO: 335) was synthesized by IDT (Coralville, Iowa). The oligo probes were 5′ end-labeled with T4 polynucleotide kinase with (γ-³²P) ATP (Promega). Twenty micrograms of total RNA from human Promyelocytic Leukemia HL-60 cells, A549, HeLa, HEK293, T47D, HepG2, T-24, MCF7, and Jurkat cells was fractionated by electrophoresis through 15% acrylamide urea gels using a TBE buffer system (Invitrogen). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by electroblotting in an Xcell SureLock™ Minicell (Invitrogen). Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using Rapid Hyb buffer solution (Amersham) using manufacturer's recommendations for oligonucleotide probes. To normalize for variations in loading and transfer efficiency membranes are stripped and probed for U6 RNA. High levels of expression of mir-21 were observed in A549 and HeLa cells; in fact, levels of mir-21 expression were noted to be among the highest of any of the miRNAs observed in HeLa cells.

Example 27 Reporter Systems for Assaying Activity of Oligomeric Compounds Targeting or Mimicking miRNAs

Reporter systems have been developed herein to assess the ability of miRNA mimics to provoke a gene silencing response and to assess whether antisense oligomeric compounds targeting miRNAs can inhibit gene silencing activity. The T-REx™-HeLa cell line (Invitrogen Corp., Carlsbad, Calif.) was used for either stable or transient transfections with plasmids constitutively expressing miRNAs, pre-miRNAs, pri-miRNAs or mimics thereof, and, in some cases, antisense oligomeric compounds targeting the expressed miRNA were also transfected into the cells. It is understood that other mammalian cells lines can also be used in this reporter system. T-REx™-HeLa cells were routinely cultured in DMEM, high glucose (Invitrogen Corporation, Carlsbad, Calif.), supplemented with 10% fetal bovine serum (Invitrogen Corporation). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were harvested when they reached 90% confluence, and on the day before transfection with expression or reporter plasmids (described in detail below), the T-REx™-HeLa cells were seeded onto 24-well plates at 50,000 cells/well. The following day, cells were transfected according to standard published procedures with various combinations of plasmids using 2 μg Lipofectamine™ 2000 Reagent (Invitrogen) per μg of plasmid DNA. When transfecting oligomeric compounds, 1-3 μg of Lipofectamine™ 2000 Reagent was used per 100 nM oligomeric compound.

Plasmids used are as follows: The pcDNA3.1©/NT-GFP (Invitrogen) plasmid, containing a CMV promoter controlling expression of a GFP reporter sequence at the N-terminus of the transcription start site was used as a control plasmid. The pcDNA3.1©/NT-GFP-mir-143 sensor plasmid contains (in addition to the elements above) three 22-nucleotide sites encoding the mir-143 miRNA binding site, downstream of the GFP coding sequence and upstream of the polyadenylation signal. The pCR3-pri-mir-143 plasmid (“pri-mir-143”) is a CMV promoter-driven constitutive expression plasmid which expresses the 110-nucleotide mir-143 pri-miRNA sequence (SEQ ID NO: 38), to act as a mir-143 pri-miRNA mimic. The pCR3-pri-mir control (“pri-mir-control”) is a CMV promoter-driven constitutive expression plasmid which is designed to express a similar 110-nucleotide pri-miRNA sequence (AGCAGCGCAGCGCCCTGTCTCCCAGCCAAGGTGGAACCTTCTGGGA AGCGGTCAGTTGGGAGTCCCTTCCCTGAAGGTTCCTCCTTGGAAGAGAGAAGTTGTTCTG CAGC; SEQ ID NO: 862) wherein the mature mir-143 sequence has been replaced with an unrelated sequence and the predicted complementary strand opposite it within the pri-miRNA structure is replaced with a nearly complementary sequence in order to preserve the stem loop as well as the bulge structure of the natural mir-143 pri-miRNA. Additionally, in order to test the effect of an oligomeric compound targeting a miRNA, the T-REx™-HeLa cells were also transfected with the uniform 2′-MOE phosphorothioate (PS) antisense oligomeric compound ISIS Number 327901 (SEQ ID NO: 319), targeted to mir-143 previously described.

Twenty-four hours post-transfection, cells were trypsinized and GFP fluorescence was analyzed by flow cytometry. Results are shown in Table 37.

TABLE 37 Mean GFP fluorescence after transfection of T-REx ™-HeLa cells Treatment pri-mir GFP GFP mir-143 327901 Mean control pri-mir-143 control sensor oligo fluorescence − − − − − 2.2 + − − − − 2.7 − + − − − 2.6 − − + − − 7.9 + − + − − 22.7 − + + − − 9.6 − − − + − 12.4 + − − + − 21.8 − + − + − 5.3 − + − + + 4.1 − − − + + 4.2 − + − − + 3.7

Plus signs, “+”, indicate the presence of the expression plasmid or oligomeric construct in transfectants; minus signs “−”, indicate the absence of same. Mean fluorescence is measured in arbitrary units.

In cells transfected with the sensor plasmid and expressing the mir-143 pri-miRNA mimic from the pCR3-pri-mir-143 plasmid, the mir-143 miRNA is expected to be processed endogenously, allowing it to bind as a mature miRNA to the RNA transcript encoding GFP and containing the mir-143 binding sites expressed from the reporter plasmid, resulting in cleavage of the reporter transcript and a decrease in fluorescence as compared to the control plasmid. From the data shown in Table 37, it was observed that expression of the pCR3-pri-mir-143 plasmid results in an inhibition of expression of GFP indicated by a decrease in fluorescence produced by the pcDNA3.1/NT-GFP-mir-143 sensor plasmid, whereas expression of the pCR3-pri-mir control plasmid had no effect on GFP reporter expression. Neither the pCR3-pri-mir control nor the pCR3-pri-mir-143 plasmid had any inhibitory effect on GFP expression from the pcDNA3.1©/NT-GFP control plasmid. Thus, the mir-143 pri-miRNA mimic oligomeric compound silences the expression of RNA transcribed from a reporter plasmid containing mir-143 target sites.

In a further study, T-REx™-HeLa cells transfected with the pcDNA3.1/NT-GFP-mir-143 sensor plasmid were treated at various dosages with the following oligomeric compounds: 1) a double-stranded RNA oligomeric compound (“ds-mir-143”) composed of ISIS Number 342199 (TGAGATGAAGCACTGTAGCTCA; SEQ ID NO: 220) representing the mir-143 sense sequence, hybridized to its perfect complement ISIS Number 342200 (TGAGCTACAGTGCTTCATCTCA; SEQ ID NO: 319); 2) a negative control dsRNA (“ds-Control”), representing a 10-base mismatched sequence antisense to the unrelated PTP1B mRNA, composed of ISIS Number 342427 (CCTTCCCTGAAGGTTCCTCC; SEQ ID NO: 863) hybridized to its perfect complement ISIS Number 342430 (GGAGGAACCTTCAGGGAAGG; SEQ ID NO: 864); 3) the pCR3-pri-mir-143 expression plasmid (“pCR3-pri-mir-143”) which expresses the 110-nucleotide mir-143 pri-miRNA; 4) the pCR3-pri-mir control (“pri-mir-control”); 5) an in vitro transcribed RNA oligomeric compound (“hairpin mir-143”) representing the 110 bp fragment of the mir-143 pri-miRNA molecule (SEQ ID NO: 38) plus an additional two cytosine nucleobases from the T7 promoter at the 5′ end; and 6) an in vitro transcribed RNA oligomeric compound (“hairpin control”) (SEQ ID NO: 862) representing a similar hairpin structure except that the mature mir-143 sequence and its complementary sequence within the pri-miRNA hairpin structure were replaced with sequences unrelated to mir-143. The RNA hairpin oligomeric compounds were in vitro transcribed using the MAXIscript Kit (Ambion Inc., Austin, Tex.) according to the manufacturer's protocol, beginning with a DNA template amplified by PCR. GFP fluorescence of treated cells was assessed using the methods described above, and it was observed that the ds-mir-143 oligomeric compound mimic inhibited expression of GFP from the sensor plasmid in a dose dependent manner. In a further embodiment, pcDNA3.1©/NT-GFP-mir-143 sensor-expressing cells treated with 20 nM mir-143 dsRNA oligomeric compound were additionally treated with 4-, 20- or 100 nM uniform 2′-MOE oligomeric compound ISIS Number 327901 (SEQ ID NO: 319), or 4-, 20- or 100 nM uniform 2′-MOE scrambled mir-143 control ISIS Number 342673 (SEQ ID NO: 758) to assess the ability of compounds to inhibit the inhibitory effect of the mir-143 dsRNA mimic. At all three concentrations, the oligomeric compound ISIS Number 327901 targeting mir-143 blocked the inhibitory effect of the mir-143 dsRNA oligomeric compound, exhibited as a recovery of GFP fluorescence.

In one embodiment, an expression system based on the pGL3-Control (Promega Corp., Madison Wis.) vector containing a CMV promoter controlling expression of a luciferase reporter sequence was used in transient transfections of HeLa cells with plasmids expressing miRNA or pri-miRNA mimics. To assess the ability of miRNA mimics to bind and regulate the expression of the luciferase reporter gene, two reporter plasmids were constructed: 1) a synthetic DNA fragment comprising two sites perfectly complementary to mir-143 were inserted into the pGL3-Control luciferase reporter vector, to create the pGL3-mir-143 sensor plasmid, and 2) a DNA fragment comprising the 3′-UTR of the LIM domain only 4 (LMO4) gene (predicted to be regulated by mir-143) was inserted into pGL3-Control to create pGL3-LMO4; this fragment was PCR-amplified using a primer beginning at position 1261 of the LMO4 sequence (GenBank Accession NM_(—)006769.2, incorporated herein as SEQ ID: 809) and the downstream primer hybridizing to the poly-A tail. In each of these plasmids, the target site was placed downstream of the luciferase coding sequence and upstream of the polyadenylation signal in the 3′-UTR of the luciferase reporter vector. The unmodified pGL3-Control luciferase reporter vector was used as a control.

HeLa cells were routinely cultured and passaged as described, and on the day before transfection with expression or reporter plasmids, the HeLa cells were seeded onto 24-well plates 50,000 cells/well. Cells were transfected according to standard published procedures with various combinations of plasmids using 2 μg Lipofectamine™ 2000 Reagent (Invitrogen) per μg of plasmid DNA, or, when transfecting oligomeric compounds, 1.25 μg of Lipofectamine™ 2000 Reagent per 100 nM oligonucleotide or double-stranded RNA. The luciferase signal in each well was normalized to the Renilla luciferase (RL) activity produced from a co-transfected plasmid, pRL-CMV, which was transfected at 0.5 μg per well. Cells were treated at various dosages (4 nM, 20 nM, and 100 nM) with the following oligomeric compound mimics: 1) “ds-mir-143,” 2) “ds-Control,” 3) “pCR3-pri-mir-143,” or 4) “pri-mir-control,” as described supra. In accordance with methods described in Example 12, supra, a luciferase assay was performed 48-hours after transfection. Briefly, cells were lysed in passive lysis buffer (PLB; Promega), and 20 ul of the lysate was then assayed for RL activity using a Dual Luciferase Assay kit (Promega) according to the manufacturer's protocol. The results below are an average of three trials and are presented as percent pGL3-Control luciferase expression normalized to pRL-CMV expression (RL). The data are shown in Table 38.

TABLE 38 Luciferase assays showing effects of oilgomeric compounds mimicking mir-143 luciferase expression (% lucif.only control) pGL3-mir-143 treatment pGL3-Control sensor pGL3-LMO4 no luciferase (pRL) 0.3 0.3 0.4 luciferase (pRL) only 100.0 101.0 100.0 ds-mir-143 (4 nM) 101.5 14.5 151.6 ds-mir-143 (20 nM) 123.8 8.0 140.1 ds-mir-143 (100 nM) 131.8 7.1 128.4 ds-Control (4 nM) 133.6 144.5 172.4 ds-Control (20 nM) 126.1 169.8 151.6 ds-Control (100 nM) 123.0 151.3 151.5 pCR3-pri-mir-143 75.6 58.6 101.9 (0.25 ug) pCR3-pri-mir-143 76.6 50.7 95.7 precursor (0.5 ug) pCR3-pri-mir-143 (1 ug) 64.7 35.0 82.5 pri-mir control (0.25 ug) 90.3 78.3 114.8 pri-mir control (0.5 ug) 57.3 61.8 95.4 pri-mir control (1 ug) 67.9 64.9 74.8

From these data, it was observed that the mir-143 dsRNA oligomeric compound and the mir-143 pri-miRNA mimic expressed from the pCR3-pri-mir-143 expression plasmid both inhibited luciferase activity from the pGL3-mir-143 sensor plasmid in a dose-dependent manner.

In another embodiment, HeLa cells were transfected with 0.03 μg pGL3-mir-143 sensor plasmid and 0.01 μg pRL-CMV plasmid, and, in addition, (except those samples described below as “without mir-143 pri-miRNA,”) were also transfected with 0.01 μg of an expression plasmid designed to express a mir-143 pri-miRNA mimic comprising a larger 430-nt fragment of the mir-143 primary miRNA transcript, referred to as “pCR3-pri-mir-143 (430)” (AGGTTTGGTCCTGGGTGCTCAAATGGCAGGCCACAGACAGGAAACACAG TTGTGAGGAATTACAACAGCCTCCCGGCCAGAGCTGGAGAGGTGGAGCCCAGGTCCCCT CTAACACCCCTTCTCCTGGCCAGGTTGGAGTCCCGCCACAGGCCACCAGAGCGGAGCAG CGCAGCGCCCTGTCTCCCAGCCTGAGGTGCAGTGCTGCATCTCTGGTCAGTTGGGAGTCT GAGATGAAGCACTGTAGCTCAGGAAGAGAGAAGTTGTTCTGCAGCCATCAGCCTGGAAG TGGTAAGTGCTGGGGGGTTGTGGGGGGCCATAACAGGAAGGACAGAGTGTTTCCAGACT CCATACTATCAGCCACTTGTGATGCTGGGGAAGTTCCTCTACACAAGTTCCCCTGGTGCC ACGATCTGCTTCACGAGTCTGGGCA; SEQ ID NO: 871). It was observed that the mir-143 pri-miRNA mimic expressed by pCR3-pri-mir-143 (430) inhibits luciferase expression from the pGL3-mir-143 sensor plasmid. To further evaluate the ability of the mir-143 pri-miRNA mimic to inhibit luciferase activity from the sensor plasmid, and to assess the ability of oligomeric compounds to interfere with the inhibition of pGL3-mir-143 sensor luciferase expression by the mir-143 pri-miRNA mimic, pGL3-mir-143 sensor-expressing HeLa cells treated with pCR3-pri-mir-143 (430) were additionally treated with varying concentrations (0-, 6.7- or 20 nM) of the following oligomeric compounds: 1) ISIS Number 327901 (SEQ ID NO: 319), a uniform 2′-MOE oligomeric compound targeting mir-143; 2) ISIS Number 342673 (SEQ ID NO: 758), a uniform 2′-MOE scrambled control; or 3) ISIS Number 327924 (SEQ ID NO: 342) targeting an unrelated miRNA (mir-129-2). ISIS Numbers 342673 and 327924 were used as negative controls. HeLa cells transfected with the pRL-CMV and pGL3-mir-143 sensor plasmids, but not treated with the pCR3-pri-mir-143 (430) hairpin precursor served as a control. In this experiment, the luciferase assay was performed 24-hours after transfection. The data are presented in Table 39 as relative luciferase activity (normalized to RL expression levels). Where present, “N.D.” indicates “no data.”

TABLE 39 Effects of oligomeric compounds on mir-143 pri-miRNA mimic-mediated inhibition of luciferase expression Relative luciferase activity SEQ ID Dose of oligomeric compound Treatment NO 0 nM 6.7 nM 20 nM 327901 319 0.97 4.0 6.4 342673 758 0.97 1.3 1.5 negative control 327924 342 0.97 0.8 1.2 negative control without pCR3-pri-mir-143 (430) N/A 13.8 N.D. N.D.

From these data, it was observed that the oligomeric compound ISIS Number 327901 targeting mir-143 blocked the inhibitory effect of the mir-143 pri-miRNA mimic, exhibited as a 4- to 6.4-fold recovery of luciferase activity in HeLa cells expressing the pGL3-mir-143 sensor plasmid.

More than four-hundred target genes have been predicted to be regulated by miRNA binding to the 3′-UTR regions of the mRNA transcript (Lewis et al., Cell, 2003, 115, 787-798). For example, at least six genes have been reported to bear regulatory sequences in their 3′-UTRs which are predicted to be bound by the mir-143 miRNA; these include the inwardly rectifying potassium channel Kir2.2 (GenBank Accession AB074970, incorporated herein as SEQ ID NO: 872), synaptotagmin III (GenBank Accession BC028379, incorporated herein as SEQ ID NO: 873), mitogen-activated protein kinase 7/extracellular signal-regulated kinase 5 (ERK5) (GenBank Accession NM_(—)139032.1, SEQ ID NO: 861), protein phosphatase 2 (formerly 2A), catalytic subunit, beta isoform (PPP2CB) (GenBank Accession NM_(—)004156.1, SEQ ID NO: 814), glyoxalase I (GLO1) (GenBank Accession NM_(—)006708.1, SEQ ID NO: 821), and LIM domain only 4 (LMO4) (GenBank Accession NM_(—)006769.2, SEQ ID NO: 809). It should be noted that one third of miRNA targets predicted in the study by Lewis, et al. are expected to be false positives (Lewis et al., Cell, 2003, 115, 787-798).

Because the present inventors independently identified the PPP2CB and GLO1 genes as potential targets of mir-143 by the RACE-PCR methods as described in Example 20, these targets were selected for further study. In addition, and described in Example 25, a novel mir-143 binding site (and, thus, a potential regulatory site) was identified within the coding sequence of the ERK5 gene; this predicted mir-143 binding site within the ERK5 coding sequence was also tested in these reporter systems.

In some embodiments, an expression system based on the pGL3-Control (Promega Corp., Madison Wis.) reporter vector and comprising predicted miRNA binding sites was used in stable transfections of HeLa cells, selecting for cells that have integrated the reporter plasmid into their genome. Because pGL3-based reporter vectors have no selectable marker for antibiotic resistance, a neomycin-resistance (Genetecin) gene was cloned into the pCR2 plasmid (Invitrogen Life Technologies, Carlsbad, Calif.) to create the pCR2-neo plasmid, and pCR2-neo was co-transfected into HeLa cells with the pGL3-mir-143-sensor plasmid at a ratio of one pCR2-neo plasmid to ten pGL3-mir-143-sensor plasmids. Co-transfected cells were then selected for the presence of the Genetecin marker and assayed for luciferase activity; Genetecin-resistant cells are very likely to have also integrated the luciferase reporter into their genome.

Establishment of Stably-Transfected Cells:

One day prior to transfection, approximately 750,000 HeLa cells are seeded onto a 10 cm dish or T-75 flask and grown in complete medium overnight at 37° C. The next day, 10 μg of pGL3-mir-143-sensor plasmid and 1 μg pCR2-neo are mixed in 2 ml OPTI-MEM™ (Invitrogen Corporation, Carlsbad, Calif.). (Linearization of circular plasmids by digestion with restriction enzyme may increase the number of stable transfectants per μg transforming DNA, but is not an essential step). 10 μl LIPOFECTIN™ reagent (Invitrogen Corporation, Carlsbad, Calif.) is mixed with 2 ml OPTI-MEM™. The plasmid/OPTI-MEM™ and OPTI-MEM™/LIPOFECTIN™ mixtures are then mixed together, and an additional 11 ml OPTI-MEM™ is added, and the resulting 15 ml cocktail is added to the cells. Cells are incubated in the plasmid/OPTI-MEM™/LIPOFECTIN™ cocktail for approximately 4 hours at 37° C., after which the cocktail is removed and replaced with fresh complete medium. The following day, cells are trypsinized and transferred to a T-175 flask. Media containing the selection agent, 500 μg/ml G418 (Geneticin; GIBCO/Life Technologies, Gaithersburg, Md.), is added and cells are grown at 37° C. Cells are re-fed daily with fresh media containing the selection agent until the majority of the cells appear to have died off and isolated colonies of neomycin-resistant cells appear. In cases where subcloning is desired, selected neomycin-resistant cells are trypsinized and plated at a concentration of 0.5 cells/well in 96-well plates, maintaining the cells in 500 μg/ml G418 selection media.

In one embodiment, five stably-transfected, neomycin-resistant, luciferase-positive, pGL3-mir-143-sensor cell clones were isolated, subcloned and selected for further testing with oligomeric compounds of the present invention. Cells stably expressing the luciferase reporter and comprising one or more miRNA binding sites were then transfected with oligomeric compounds mimicking miRNAs, pre-miRNAs or pri-miRNAs in order to assess the ability of these miRNA mimics to bind and regulate the expression of the luciferase reporter.

An expression system based on the pGL3-Control (Promega Corp., Madison Wis.) reporter vector and comprising predicted miRNA binding sites was used in transient transfections of HeLa cells with plasmids expressing oligomeric compounds mimicking miRNAs, pre-miRNAs or pri-miRNAs in order to assess the ability of these miRNA mimics to bind and regulate the expression of the luciferase reporter. The effect of increasing the copy number of the miRNA-binding site in the target was also tested by including multiple binding sites in artificial reporter constructs. It is understood that the presence of multiple miRNA-binding sites in a target can include binding sites for different miRNAs.

The following reporter plasmids were constructed by cloning the specified fragment into the XbaI site of the pGL3-control plasmid, placing the potential miRNA-binding site in the 3′-UTR of the luciferase reporter: The reporter plasmid pGL3-bulge(×3) contains three contiguous copies of the sequence (TGAGCTACAGCTTCATCTCA; herein incorporated as SEQ ID NO: 874) which represents a sequence complementary to the mir-143 miRNA except that it is missing 2 nucleotides such that the mir-143 miRNA is presumed to adopt a bulged structure when it hybridizes to this target sequence. The pGL3-GLO1 reporter plasmid contains a DNA fragment comprising the 3′-UTR of the GLO1 sequence; this fragment was PCR-amplified using a primer beginning at nucleotide number 621 of the GLO1 sequence (GenBank Accession NM_(—)006708.1, SEQ ID NO: 821) and the downstream primer hybridizing to the poly A tail. The pGL3-PP2A reporter plasmid contains a DNA fragment comprising the 3′-UTR of the PP2A gene; this fragment was PCR-amplified using a primer beginning at nucleotide number 921 of the PP2A sequence (GenBank Accession NM_(—)004156.1) and the downstream primer hybridizing to the poly A tail. The reporter plasmid pGL3-ERK5-3′-UTR(×1) contains one copy of the sequence TATTCTGCAGGTTCATCTCAG (herein incorporated as SEQ ID NO: 875), found in the 3′-UTR of ERK5 and predicted by Lewis, et al. to be bound by the mir-143 miRNA, and the reporter plasmid pGL3-ERK5-3′UTR(×3) has three contiguous copies of this sequence. The reporter plasmid pGL3-ERK5-3′UTR(ext) contains one copy of the sequence CGGCTTGGATTATTCTGCAGGTTCATCTCAGACCCACCTTT (herein incorporated as SEQ ID NO: 876), which includes an additional ten nucleotides at either end of the mir-143 binding site in 3′-UTR of ERK5 predicted by Lewis, et al. (Lewis et al., Cell, 2003, 115, 787-798). The plasmids pGL3-ERK5-cds(×1), pGL3-ERK5-cds(×2), pGL3-ERK5-cds(×3), and pGL3-ERK5-cds(×5) contain one, two, three or five contiguous copies, respectively, of the novel mir-143 binding site (TTGAGCCCAGCGCTCGCATCTCA; herein incorporated as SEQ ID NO: 877) we identified within the coding sequence of ERK5. The unmodified pGL3-Control luciferase reporter vector was used as a negative control, and the pGL3-mir-143 sensor reporter plasmid was used as a positive control.

HeLa cells were routinely cultured and passaged as described. In some embodiments, HeLa cells were transfected with 0.05 μg of the relevant pGL3-sensor plasmid and 0.01 μg pRL-CMV plasmid. Additionally, in some embodiments, cells were treated at various dosages (11 nM, 33 nM, and 100 nM) with the following oligomeric compound mimics: 1) ds-mir-143, or 2) ds-Control as described. In accordance with methods described in Example 12, a luciferase assay was performed 24-hours after transfection. The results, shown in Tables 40 and 41, are an average of three trials. Data are presented as percent untreated control (luciferase plasmid only, not treated with oligomeric compound) luciferase expression, normalized to pRL-CMV levels.

TABLE 40 Effects of oligomeric compounds mimicking mir-143 on luciferase expression Reporter ds-mir-143 ds-control plasmid 11 nM 33 nM 100 nM 11 nM 33 nM 100 nM pGL3-Control 90.7 94.2 72.5 113.4 79.6 87.0 pGL3- 50.7 35.4 17.2 111.3 82.6 84.7 bulge(x3) pGL3-ERK5- 81.9 84.7 62.2 103.2 79.6 77.6 3′UTR(x1)

From these data, it was observed that, while treatment of HeLa cells expressing the reporter plasmids with the ds-control did not appear to significantly affect luciferase expression, the mir-143 dsRNA mimic compound inhibited luciferase activity from the pGL3-bulge(×3) sensor plasmid in a dose-dependent manner.

TABLE 41 Effects of oligomeric compounds mimicking mir-143 on luciferase expression ds-mir-143 ds-control 33 100 33 100 Reporter plasmid 11 nM nM nM 11 nM nM nM pGL3-Control 110.2 124.3 92.3 114.1 95.6 103.0 pGL3-mir-143 sensor 15.0 15.0 11.1 114.5 108.9 97.1 pGL3-bulge(x3) 36.1 33.9 22.2 109.5 103.2 92.4 pGL3-ERK5-3′UTR(x1) 92.2 108.1 81.9 106.2 99.6 90.1 pGL3-ERK5-3′UTR(x3) 51.7 51.0 28.2 104.6 103.4 95.7 pGL3-ERK5-cds(x1) 101.3 115.4 77.4 100.6 102.1 96.2 pGL3-ERK5-cds(x2) 92.7 113.8 63.6 111.3 99.2 90.4 pGL3-ERK5-cds(x3) 73.5 77.9 49.4 105.2 96.6 79.9 pGL3-ERK5-cds(x5) 49.4 44.5 23.9 103.0 113.4 89.9 pGL3-ERK5-3′UTR(ext) 89.0 106.7 81.4 96.8 108.9 89.4

From these data it was observed that treatment of HeLa cells expressing the pGL3-bulge(×3) reporter plasmid with the ds-mir-143 miRNA mimic oligomeric compound resulted in a dose-dependent inhibition of luciferase activity while the ds-control oligomeric compound had no effect as described previously. Treatment of HeLa cells expressing the pGL3-ERK5-3′UTR(×1) (containing one copy of the mir-143 binding site predicted by Lewis, et al.) with the ds-mir-143 mimic oligomeric compound did not inhibit luciferase activity, although increasing the number of potential mir-143 binding sites in the pGL3-ERK5-3′UTR(×3) reporter plasmid to three appeared to favor the binding of the ds-mir-143 mimic and inhibition of luciferase activity. Treatment of cells expressing the pGL3-ERK5-cds(×1) or pGL3-ERK5-cds(×2) reporter plasmids bearing a one or two copies, respectively, of the novel mir-143 binding site identified in the coding sequence of the ERK5 gene with 11- or 33 nM of the ds-mir-143 mimic oligomeric compound did not appear to inhibit luciferase activity, although treatment with 100 nM of the ds-mir-143 mimic did reduce luciferase expression. Treatment of cells expressing the pGL3-ERK5-cds(×3) or pGL3-ERK5-cds(×5) reporter plasmids, bearing three or five of copies, respectively, of the novel mir-143 binding site in the ERK5 coding sequence, with the ds-mir-143 mimic oligomeric compound resulted in a reduction in luciferase activity. The pGL3-ERK5-cds(×5) reporter plasmid exhibited a dose-responsiveness with increasing concentration of the mir-143 mimic oligomeric compound. Taken together, these results support the conclusion that multiple miRNAs and miRNA binding sites may cooperate to silence gene expression.

In order to assess the ability of miRNAs to bind predicted miRNA binding sites and regulate the expression of the luciferase reporter, in some embodiments, expression systems based on the pGL3-Control (Promega Corp., Madison Wis.) reporter vector and comprising either a mir-15a, mir-21, or a mir-23b miRNA binding site were developed and used in transient transfections of HeLa cells to determine whether the endogenous mir-15a, mir-21, or mir-23b miRNAs, respectively, could repress luciferase reporter gene expression.

The pGL3-mir-15a sensor plasmid was created by cloning the sequence (CACAAACCATTATGTGCTGCTA; SEQ ID NO: 369), complementary to the mir-15a miRNA, into the Xba site of the pGL3-Control plasmid, placing the potential miRNA-binding site in the 3′UTR of the luciferase reporter. This reporter plasmid was used to transfect HeLa cells and it was observed that the endogenous mir-15a miRNA was able to inhibit luciferase expression from the pGL3-mir-15a sensor plasmid. Thus, to further evaluate the ability of the mir-15a miRNA to bind this target site encoded by the pGL3-mir-15a sensor plasmid, and to assess the ability of oligomeric compounds to interfere with mir-15a-mediated silencing, pGL3-mir-15a sensor-expressing HeLa cells were treated with varying concentrations (3-, 10- or 30 nM) of the following oligomeric compounds: ISIS Number 327951 (SEQ ID NO: 369) is a uniform 2′-MOE compound targeting the mature mir-15a-1 miRNA. ISIS Numbers 356213 (SEQ ID NO: 878), 356215 (SEQ ID NO: 879), 356216 (SEQ ID NO: 880), 356218 (SEQ ID NO: 881), 356221 (SEQ ID NO: 882), 356227 (SEQ ID NO: 883) and 356229 (SEQ ID NO: 884) are phosphorothioate, uniform 2′-MOE oligomeric compounds designed and synthesized to target the entire length of the mir-15a pri-miRNA molecule (described in detail in Example 28, below). The uniform 2′-MOE phosphorothioate oligomeric compounds ISIS Number 327901 (SEQ ID NO: 319), targeting an unrelated miRNA (mir-143) and ISIS Number 342673 (AGACTAGCGGTATCTTTATCCC; herein incorporated as SEQ ID NO: 758), containing 15 mismatches with respect to the mature mir-143 miRNA, were used as negative controls. The data presented in Table 42 are the average of three trials and are presented as percent untreated control (luciferase plasmid only, not treated with oligomeric compound) luciferase expression, normalized to pRL-CMV levels.

TABLE 42 Effects of oligomeric compounds on mir-15a miRNA-mediated inhibition of luciferase expression Relative luciferase activity Dose of SEQ ID oligomeric compound Treatment NO 3 nM 10 nM 30 nM 327901 319 83.6 96.6 88.2 negative control 342673 758 104.5 82.6 85.7 negative control 327951 369 151.0 207.6 137.1 356213 878 101.2 80.5 109.9 356215 879 98.0 116.7 79.6 356216 880 102.8 84.7 113.2 356218 881 91.6 110.3 85.7 356221 882 106.8 74.0 81.2 356227 883 86.1 117.8 101.5 356229 884 109.7 100.3 97.5

From these data, it was observed that the oligomeric compound ISIS Number 327951 targeting the mature mir-15a miRNA blocked the inhibitory effect of mir-15a, exhibited as a recovery and increase in luciferase activity in HeLa cells expressing the pGL3-mir-15a sensor plasmid.

The pGL3-mir-23b sensor plasmid was created by cloning the sequence (GTGGTAATCCCTGGCAATGTGAT; SEQ ID NO: 307), representing a sequence complementary to the mir-23b miRNA, into the Xba site of the pGL3-Control plasmid, placing the potential miRNA-binding site in the 3′UTR of the luciferase reporter. This reporter plasmid was used to transfect HeLa cells and it was observed that the endogenous mir-23b miRNA was able to inhibit luciferase expression from the pGL3-mir-23b sensor plasmid. Thus, to further evaluate the ability of the mir-23b miRNA to bind this target site encoded by the pGL3-mir-23b sensor plasmid, and to assess the ability of oligomeric compounds to interfere with mir-23b-mediated silencing, pGL3-mir-23b sensor-expressing HeLa cells were treated with varying concentrations (1.3-, 5- or 20 nM) of the following oligomeric compounds: ISIS Number 327889 (SEQ ID NO: 307), a phosphorothioate uniform 2′-MOE oligomeric compound, and ISIS Number 340925 (SEQ ID NO: 307), a 2′-MOE 5-10-8 gapmer oligomeric compound, both targeting mir-23b. The uniform 2′-MOE phosphorothioate oligomeric compound ISIS Number 327924 (SEQ ID NO: 342) targeting an unrelated miRNA (mir-129-2) was used as a negative control. The data are the average of three trials, and are presented in Table 43 as relative luciferase activity (normalized to pRL-CMV luciferase plasmid only, not treated with oligomeric compound).

TABLE 43 Effects of oligomeric compounds on mir-23b miRNA-mediated inhibition of luciferase expression Fold change luciferase Dose of SEQ ID oligomeric compound Treatment NO 1.3 nM 5 nM 20 nM 327924 342 1.15 0.68 0.92 negative control 327889-uniform MOE 307 3.75 3.46 7.40 340925-gapmer 307 0.99 1.41 1.19

From these data, it was observed that, at all doses, ISIS Number 327889, the uniform 2′-MOE oligomeric compound targeting the mature mir-23b miRNA, de-repressed the expression of the luciferase reporter. Thus, ISIS 327889 reversed the silencing effect of the mir-23b miRNA, apparently by inhibiting the binding of mir-23b to its target site encoded by the pGL3-mir-23b sensor plasmid.

The pGL3-mir-21 sensor plasmid was created by cloning the sequence (TCAACATCAGTCTGATAAGCTA; SEQ ID NO: 335), representing a sequence complementary to the mir-21 miRNA, into the Xba site of the pGL3-Control plasmid, placing the potential miRNA-binding site in the 3′UTR of the luciferase reporter. This reporter plasmid was used to transfect HeLa cells and it was observed that the endogenous mir-21 miRNA was able to inhibit luciferase expression from the pGL3-mir-21 sensor plasmid. Thus, to further evaluate the ability of the mir-21 miRNA to bind this target site encoded by the pGL3-mir-21 sensor plasmid, and to assess the ability of oligomeric compounds to interfere with mir-21-mediated silencing, pGL3-mir-21 sensor-expressing HeLa cells were treated with varying concentrations (10 nM or 50 nM) of the following oligomeric compounds: ISIS Number 327917 (SEQ ID NO: 335), a phosphorothioate uniform 2′-MOE oligomeric compound; ISIS Number 338697 (TGCCATGAGATTCAACAGTC; herein incorporated as SEQ ID NO: 524), a uniform 2′-MOE oligomeric compound targeting the mir-21 pri-miRNA molecule; and ISIS Number 328415 (SEQ ID NO: 524), a 2′-MOE 5-10-5 gapmer oligomeric compound targeting the mir-21 pri-miRNA. The uniform 2′-MOE phosphorothioate oligomeric compound ISIS Number 327901 (SEQ ID NO: 319) targeting an unrelated miRNA (mir-143) was used as a negative control. The data are the average of three trials and are presented in Table 44 as percent untreated control (luciferase plasmid only, not treated with oligomeric compound) luciferase expression, normalized to pRL-CMV levels.

TABLE 44 Effects of oligomeric compounds on mir-21 miRNA-mediated inhibition of luciferase expression % UTC Dose of oligomeric SEQ ID compound Treatment NO 10 nM 50 nM 327901 319 74.2 83.1 negative control 327917 335 1037.6 847.5 338697 524 87.0 84.8 328415 524 66.0 104.4

From these data, it was observed that, at both doses, treatment of HeLa cells with ISIS Number 327917, the uniform 2′-MOE oligomeric compound targeting the mature mir-21 miRNA, de-repressed the expression of the luciferase reporter. Thus, ISIS 327917 reversed the silencing effect of the endogenous mir-21 miRNA, apparently by inhibiting the binding of mir-21 to its target site encoded by the pGL3-mir-21 sensor plasmid.

Therefore, oligomeric compounds targeting and/or mimicking the mir-143, mir-15a, mir-23b and mir-21 miRNAs and their corresponding pri-miRNA molecules have been demonstrated to bind to target RNA transcripts and silence reporter gene expression.

Example 28 Effects of Oligomeric Compounds on Expression of Pri-miRNAs

As described above in Example 19, pri-miRNAs, often hundreds of nucleotides in length, are processed by a nuclear enzyme in the RNase III family known as Drosha, into approximately 70 nucleotide-long pre-miRNAs (also known as stem-loop structures, hairpins, pre-mirs or foldback miRNA precursors), and pre-miRNAs are subsequently exported from the nucleus to the cytoplasm, where they are processed by human Dicer into double-stranded miRNAs, which are subsequently processed by the Dicer RNase into mature miRNAs. It is believed that, in processing the pri-miRNA into the pre-miRNA, the Drosha enzyme cuts the pri-miRNA at the base of the mature miRNA, leaving a 2-nt 3′overhang (Lee, et al., Nature, 2003, 425, 415-419). The 3′ two-nucleotide overhang structure, a signature of RNaseIII cleavage, has been identified as a critical specificity determinant in targeting and maintaining small RNAs in the RNA interference pathway (Murchison, et al., Curr. Opin. Cell Biol., 2004, 16, 223-9).

The oligomeric compounds of the present invention are believed to disrupt pri-miRNA and/or pre-miRNA structures, and sterically hinder Drosha and/or Dicer cleavage, respectively. Additionally, oligomeric compounds capable of binding to the mature miRNA are believed to prevent the RISC-mediated binding of a miRNA to its mRNA target, either by cleavage or steric occlusion of the miRNA.

Using the real-time RT-PCR methods described in Example 19, the expression levels of the mir-15a pri-miRNA were compared in HepG2 cells treated with a nested series of chimeric gapmer oligomeric compounds, targeting and spanning the entire length of the mir-15a pri-miRNA; these compounds are shown in Table 45, below. Each gapmer is 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.” The wings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. Using the transfection methods described herein, HepG2 cells were treated with 100 nM of each of these gapmer oligomeric compounds. Total RNA was isolated from HepG2 cells by lysing cells in 1 mL TRIZOL™ (Invitrogen) using the manufacturer's recommended protocols. Real-time RT-PCR analysis was performed using a primer/probe set specific for the mir-15a pri-miRNA molecule to assess the effects of these compounds on expression of the mir-15a pri-miRNA molecule. ISIS 339317 (GTGTGTTTAAAAAAAATAAAACCTTGGA; SEQ ID NO.: 885) was used as the forward primer, ISIS 339318 (TGGCCTGCACCTTTTCAAA; SEQ ID NO.: 886) was used as the reverse primer, and ISIS 339319 (AAAGTAGCAGCACATAATGGTTTGTGG; SEQ ID NO.: 887) was used as the probe. Total RNA was quantified using RiboGreen™ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.), expression levels observed for each target are normalized to 5.8S rRNA, and values are expressed relative to the untreated control. Inhibition of expression of the mir15a pri-miRNA by these gapmer oligomeric compounds is expressed as a percentage of RNA levels in untreated control cells. Results of these experiments are described in Table 45 below:

TABLE 45 Effects of chimeric oligomeric compounds on expression of the mir-15a pri-miRNA SEQ Expression of ISIS ID mir-15a pri-miRNA Number NO Sequence (% UTC) 347964 878 TATAACATTGATGTAATATG 13.7 347965 888 GCTACTTTACTCCAAGGTTT 86.0 347966 879 TGCTACTTTACTCCAAGGTT 39.2 347967 880 GCACCTTTTCAAAATCCACA 152.3 347968 889 CCTGCACCTTTTCAAAATCC 8.4 347969 881 TGGCCTGCACCTTTTCAAAA 39.5 347970 890 ATATGGCCTGCACCTTTTCA 2.2 347971 891 ACAATATGGCCTGCACCTTT 92.8 347972 882 AGCACAATATGGCCTGCACC 98.6 347973 892 GGCAGCACAATATGGCCTGC 143.3 347974 893 TGAGGCAGCACAATATGGCC 98.1 347975 894 TTTTGAGGCAGCACAATATG 9.2 347976 895 TATTTTTGAGGCAGCACAAT 73.0 347977 896 TTGTATTTTTGAGGCAGCAC 111.3 347978 883 TCCTTGTATTTTTGAGGCAG 51.1 347979 897 AGATCCTTGTATTTTTGAGG 74.9 347980 884 AGATCAGATCCTTGTATTTT 3.6 347981 898 AGAAGATCAGATCCTTGTAT N/D 347982 899 TTCAGAAGATCAGATCCTTG 82.2 347983 900 AAATATATTTTCTTCAGAAG 13.0

From these data, it was observed that oligomeric compounds ISIS Numbers 347964, 347966, 347968, 347970, 347975, 347980 and 347983 show significant inhibition of expression of the mir-15a pri-miRNA molecule. Thus, it is believed that the antisense oligomeric compounds ISIS Numbers 347964, 347966, 347968, 347970, 347975, 347980 and 347983 bind to the mir-15a pri-miRNA and/or pre-miRNA molecules and cause their degradation and cleavage.

From these data, it was observed that oligomeric compounds ISIS Numbers 347967, 347977 and 347973 stimulate an increase in expression levels of the mir-15a pri-miRNA. It is believed that the oligomeric compounds ISIS Numbers 347967, 347977 and 347973 bind to the mir-15a pri-miRNA and inhibit its processing into the mature mir-15a miRNA. It is believed that, in addition to the increase in the levels of the mir-15a pri-miRNA observed upon treatment of cells with the oligomeric compounds ISIS Numbers 347977, 347967 and 347973, a drop in expression levels of the fully processed mature mir-15a miRNA may also trigger a feedback mechanism signaling these cells to increase production of the mir-15a pri-miRNA.

The gapmer oligomeric compounds targeting the mir-15b and mir-15-a-1 mature miRNAs described above were also transfected into T47D cells according to standard procedures. In addition, uniform 2′-MOE and 2′-MOE gapmer oligomeric compounds targeting the mature mir-15a-1 and mir-15b miRNAs were also transfected into T47D cells, for analysis of their effects on mir-15a-1 and mir-15b pri-miRNA levels. The oligomeric compounds ISIS Number 327927 (SEQ ID NO: 345), a uniform 2′-MOE compound and ISIS Number 345391 (SEQ ID NO: 345), a 2′-MOE 5-10-7 gapmer compound, both target mir-15b. The oligomeric compounds ISIS Number 327951 (SEQ ID NO: 369), a uniform 2′-MOE compound, and ISIS Number 345411 (SEQ ID NO: 369), a 2′-MOE 5-10-7 gapmer compound, both target mir-15a-1. Oligomeric compounds ISIS Number 129686 (CGTTATTAACCTCCGTTGAA; SEQ ID NO: 901), and ISIS Number 129691 (ATGCATACTACGAAAGGCCG; SEQ ID NO:902), both universal scrambled controls, as well as ISIS Number 116847 (CTGCTAGCCTCTGGATTTGA; SEQ ID NO: 844) targeting an unrelated gene, PTEN, were used as negative controls. ISIS Numbers 129686, 129691, and 116847 are phosphorothiated 2′-MOE 5-10-5 gapmers, and all cytosines are 5-methylcytosines. T47D cells (seeded in 12-well plates) were treated with these oligomeric compounds, and RNA was isolated from the treated cells by lysing in 1 mL TRIZOL™ (Invitrogen) and total RNA was prepared using the manufacturer's recommended protocols. To assess the effects of these compounds on expression of the mir-15a or mir-15b pri-miRNA molecules, real-time RT-PCR analysis was performed using either the primer/probe set specific for the mir-15a pri-miRNA molecule described above, or a primer probe set specific for the mir-15b pri-miRNA molecule: ISIS 339320 (CCTACATTTTTGAGGCCTTAAAGTACTG; SEQ ID NO: 903) was used as the forward primer for the mir-15b pri-miRNA, ISIS 339321 (CAAATAATGATTCGCATCTTG ACTGT; SEQ ID NO: 904) was used as the reverse primer for the mir-15b pri-miRNA, and ISIS 339322 (AGCAGCACATCATGGTTTACATGC; SEQ ID NO: 905) was used as the probe. Total RNA was quantified using RiboGreen™ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.), expression levels observed for each target were normalized to 5.8S rRNA, and values are expressed relative to the untreated control. Inhibition of expression of the mir15a or mir-15b pri-miRNA molecules upon treatment with these oligomeric compounds is was assessed and expressed as a percentage of RNA levels in untreated control cells.

On multiple repeats of these experiments, it was observed that the uniform 2′-MOE oligomeric compounds ISIS Number 327927 (SEQ ID NO: 345) and ISIS Number 327951 (SEQ ID NO: 369), targeted to the mature mir-15b and mir-15a-1 miRNAs, respectively, each stimulate an approximately 2.5- to 3.5-fold increase in expression of the mir-15a pri-miRNA molecule and an approximately 1.5- to 2.5-fold increase in the expression of the mir-15b pri-miRNA molecule. Therefore, it is believed that ISIS Numbers 327927 and 327951 can bind to the mir-15a and/or mir-15b pri-miRNA or pre-miRNA molecules and interfere with their processing into the mature mir-15a or mir-15b miRNAs. It is also recognized that a decrease in levels of the mature, processed forms of the mir-15a or mir-15b miRNAs in T47D cells treated with ISIS Number 345411 (SEQ ID NO: 369), ISIS Number 327927 (SEQ ID NO: 345) or ISIS Number 327951 (SEQ ID NO: 369) may also trigger a feedback mechanism that signals these cells to increase production of the mir-15a and/or mir-15b pri-miRNA molecules.

In accordance with the present invention, a nested series of uniform 2′-MOE oligomeric compounds were designed and synthesized to target the entire length of the mir-15a pri-miRNA molecule. Each compound is 19 nucleotides in length, composed of 2′-methoxyethoxy (2′-MOE) nucleotides and phosphorothioate (P═S) internucleoside linkages throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds are shown in Table 46. The compounds can be analyzed for their effect on mature miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR, or they can be used in other assays to investigate the role of miRNAs or the function of targets downstream of miRNAs.

TABLE 46 Uniform 2′-MOE PS Compounds targeting the mir-15a pri-miRNA ISIS SEQ ID Number NO Sequence 356213 878 TATAACATTGATGTAATATG 356214 879 GCTACTTTACTCCAAGGTTT 356215 880 TGCTACTTTACTCCAAGGTT 356216 881 GCACCTTTTCAAAATCCACA 356217 882 CCTGCACCTTTTCAAAATCC 356218 883 TGGCCTGCACCTTTTCAAAA 356219 884 ATATGGCCTGCACCTTTTCA 356220 888 ACAATATGGCCTGCACCTTT 356221 889 AGCACAATATGGCCTGCACC 356222 890 GGCAGCACAATATGGCCTGC 356223 891 TGAGGCAGCACAATATGGCC 356224 892 TTTTGAGGCAGCACAATATG 356225 893 TATTTTTGAGGCAGCACAAT 356226 894 TTGTATTTTTGAGGCAGCAC 356227 895 TCCTTGTATTTTTGAGGCAG 356228 896 AGATCCTTGTATTTTTGAGG 356229 897 AGATCAGATCCTTGTATTTT 356230 898 AGAAGATCAGATCCTTGTAT 356231 899 TTCAGAAGATCAGATCCTTG 356232 900 AAATATATTTTCTTCAGAAG

Using the real-time RT-PCR methods described, the expression levels of the mir-15a pri-miRNA were compared in T47D cells treated with the nested series of uniform 2′-MOE oligomeric compounds, targeting and spanning the entire length of the mir-15a pri-miRNA. The region encompassing the mir-15a primary transcript (the complement of nucleotides 31603159 to 31603468 of GenBank Accession number NT_(—)024524.13; AAATAATTATG CATATTACATCAATGTTATAATGTTTAAACATAGATTTTTTTACATGCATTCTTTTTTTCCT GAAAGAAAATATTTTTTATATTCTTTAGGCGCGAATGTGTGTTTAAAAAAAATAAAACCT TGGAGTAAAGTAGCAGCACATAATGGTTTGTGGATTTTGAAAAGGTGCAGGCCATATTG TGCTGCCTCAAAAATACAAGGATCTGATCTTCTGAAGAAAATATATTTCTTTTTATTCATA GCTCTTATGATAGCAATGTCAGCAGTGCCTTAGCAGCACGTAAATATTGGCGTTAAG) is incorporated herein as SEQ ID NO: 906. ISIS Number 356215 (SEQ ID NO: 879) targets a region flanking and immediately 5′ to the predicted 5′ Drosha cleavage site in the mir-15a pri-miRNA. ISIS Number 356218 (SEQ ID NO: 881) targets a region in the loop of the mir-15a pri-miRNA. ISIS 356227 (SEQ ID NO: 883) targets a region flanking and immediately 3′ to the predicted 3′ Drosha cleavage site in the mir-15a pri-miRNA. Additionally, oligomeric compound ISIS 327951 (SEQ ID NO: 369), a uniform 2′-MOE compound targeting the mature mir-15a-1 miRNA, was tested for comparison. Oligomeric compounds ISIS 327901 (SEQ ID NO: 319) targeting the mature mir-143 miRNA; ISIS 129690, (TTAGAATACGTCGCGTTATG; SEQ ID NO: 907), a phosphorothioate 5-10-5 MOE gapmer used as a universal scrambled control; and ISIS 116847 (CTGCTAGCCTCTGGATTTGA; SEQ ID NO: 844), a uniform 5-10-5 2′-MOE gapmer targeting an unrelated gene, PTEN, were used as negative controls. Using the transfection methods previously described, T47D cells were treated with 100 nM of each of these oligomeric compounds. Total RNA was isolated by lysing cells in 1 mL TRIZOL™ (Invitrogen) using the manufacturer's recommended protocols. real-time RT-PCR analysis was performed using a primer/probe set specific for the mir-15a pri-miRNA molecule [forward primer-ISIS 339317 (SEQ ID NO.: 885), reverse primer-ISIS 339318 (SEQ ID NO.: 886), and probe=ISIS 339319 (SEQ ID NO.: 887)]. Total RNA was quantified using RiboGreen™ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.), expression levels observed for each target were normalized to 5.8S rRNA, and values were expressed relative to the untreated control (UTC). Effects on expression of the mir-15a pri-miRNA molecule resulting from treatment of T47D cells with these uniform 2′-MOE oligomeric compounds is expressed as a percentage of RNA levels in untreated control cells. Results of these experiments are described in Table 47 below:

TABLE 47 Effects of uniform 2′-MOE oligomeric compounds on mir-15a pri-miRNA expression ISIS # SEQ ID NO: target % UTC UTC N/A N/A 100 129690 XXX N/A 121 scrambled control 327901 319 mir-143 132 116847 844 PTEN mRNA 132 327951 369 mature mir-15a-1 713 356213 878 >100 bp upstream of mature mir-15a 171 356215 879 flanking 5′ Drosha cleavage site 1005 of mir-15a-1 pri-miRNA 356216 880 mir-15a-1 pri-miRNA 503 356218 881 loop of mir-15a-1 pri-miRNA 392 356221 882 mir-15a-1 pri-miRNA 444 356224 894 mir-15a-1 pri-miRNA 592 356227 883 flanking 3′ Drosha cleavage site 879 of mir-15a-1 pri-miRNA 356229 884 mir-15a-1 pri-miRNA 818 356231 899 mir-15a-1 pri-miRNA 811 356232 900 mir-15a-1 pri-miRNA 631

From these data, it was observed that the uniform 2′-MOE oligomeric compounds ISIS Numbers 327927, 327951, 356215, 356216, 356218, 356221, 356224, 356227, 356229, 356231 and 356232 stimulate an increase in levels of the mir-15a pri-miRNA molecule as detected by real-time RT-PCR. Notably, oligomeric compounds ISIS Numbers 356215 and 356227 which target the regions immediately flanking the predicted 5′ and 3′ Drosha cleavage sites in the mir-15a pri-miRNA, respectively, were observed to stimulate the greatest increases in expression of the mir-15a pri-miRNA. It is believed that these oligomeric compounds bind to the mir-15a pri-miRNA and/or pre-miRNA molecules and interfere with their processing into the mature mir-15a miRNA, possibly by interfering with the activity of RNase III-like enzymes such as human Dicer and/or Drosha. The resultant decrease in levels of the processed mature mir-15a miRNA may trigger a feedback mechanism leading to an upregulation of production of the mir-15a pri-miRNA molecule. Not mutually exclusive with the processing interference and the feedback mechanisms is the possibility that treatment with oligomeric compounds could stimulate the activity of an RNA-dependent RNA polymerase (RdRP) that amplifies the mir-15a pri-miRNA or pre-miRNA molecules. It is understood that such oligomeric compound-triggered mechanisms may be operating not only upon regulation of mir-15a production and processing, but may also be found to regulate the production and processing of other miRNAs.

The expression levels of mir-24-2, let-71, and let-7d were assessed in HeLa or T-24 cells treated with various uniform 2′-MOE oligomeric compounds targeting mature miRNAs. For example, using the transfection methods previously described, HeLa cells were treated with 100 nM of the oligomeric compound ISIS Number 327945 (SEQ ID NO: 363) targeting the mir-24-2 mature miRNA. Total RNA was isolated and expression levels of the mir-24-2 pri-miRNA were analyzed by real-time quantitative RT-PCR using a primer/probe set specific for the mir-24-2 pri-miRNA molecule [forward primer=ISIS 359358 (CCCTGGGCTCTGCCT; herein incorporated as SEQ ID NO.: 908), reverse primer-ISIS 359359 (TGTACACAAACCAACTGTGTTTC; herein incorporated as SEQ ID NO.: 909), and probe=ISIS 359360 (CGTGCCTACTGAGC; herein incorporated as SEQ ID NO.: 910)]. An approximately 35-fold increase in expression levels of the mir-24-2 pri-miRNA molecule was observed in HeLa cells treated with the oligomeric compound ISIS 327945 as detected by real-time RT-PCR.

Using the transfection methods previously described, HeLa cells were treated with 100 nM of the oligomeric compound ISIS Number 327890 (SEQ ID NO: 308) targeting the let-71 mature miRNA. Total RNA was isolated and expression levels of the let-71 pri-miRNA were analyzed by real-time quantitative RT-PCR using a primer/probe set specific for the let-71 pri-miRNA molecule [forward primer-ISIS 341684 (TGAGGTAGTAGTTTGTGCTGTTGGT; herein incorporated as SEQ ID NO.: 777), reverse primer=ISIS 341685 (AGGCAGTAGCTTGCGCAGTTA; herein incorporated as SEQ ID NO.: 778), and probe=ISIS 341686 (TTGTGACATTGCCCGCTGTGGAG; herein incorporated as SEQ ID NO.: 779)]. An approximately 4-fold increase in expression levels of the let-71 pri-miRNA molecule was observed in HeLa cells treated with the oligomeric compound ISIS 327890 as detected by real-time RT-PCR.

Using the transfection methods previously described, supra, T-24 cells were treated with 100 nM of the oligomeric compound ISIS Number 327926 (SEQ ID NO: 344) targeting the let-7d mature miRNA. Total RNA was isolated and expression levels of the let-7d pri-miRNA were analyzed by real-time quantitative RT-PCR using a primer/probe set specific for the let-7d pri-miRNA molecule (forward primer=ISIS 341678 (CCTAGGAAGAGGTAG TAGGTTGCA; herein incorporated as SEQ ID NO.: 771), reverse primer=ISIS 341679 (CAGCAGGTCGTATAGTTACCTCCTT; herein incorporated as SEQ ID NO.: 772), and probe=ISIS 341680 (AGTTTTAGGGCAGGGATTTTGCCCA; herein incorporated as SEQ ID NO.: 773)). An approximately 1.7-fold increase in expression levels of the let-7d pri-miRNA molecule was observed in T-24 cells treated with the oligomeric compound ISIS 327926 as detected by real-time RT-PCR.

Thus, treatment with uniform 2′-MOE oligomeric compounds targeting mature miRNAs appears to result in an induction of expression of the corresponding pri-miRNA molecule.

In one embodiment, the expression of mir-21 (noted to be expressed at high levels in HeLa cells) was assessed in cells treated with oligomeric compounds. Using the transfection methods previously described, HeLa cells were treated with 100 nM of the uniform 2′-MOE oligomeric compound ISIS Number 327917 (SEQ ID NO: 335) targeting the mir-21 mature miRNA. Total RNA was isolated by lysing cells in 1 mL TRIZOL™ (Invitrogen) using the manufacturer's recommended protocols. By Northern blot analysis of total RNA from HeLa cells treated with ISIS 327917, expression levels of the mir-21 mature miRNA were observed to be reduced to 50% of those of untreated control cells. Furthermore, expression levels of the mir-21 pri-miRNA were found to increase in these HeLa cells treated with the oligomeric compound ISIS 327917. Real-time RT-PCR analysis was also performed on HeLa cells treated with ISIS 327917 using a primer/probe set specific for the mir-21 pri-miRNA molecule [forward primer=ISIS 339332 (GCTGTACCACCTTGTCGGGT; herein incorporated as SEQ ID NO.: 911), reverse primer-ISIS 339333 (TCGACTGGTGTTGCCATGA; herein incorporated as SEQ ID NO.: 912), and probe=ISIS 339334 (CTTATCAGACTGATGTTGACTGTTGAAT; herein incorporated as SEQ ID NO.: 913)]. Total RNA was quantified using RiboGreen™ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.), expression levels observed for the target were normalized to 5.8S rRNA, and values were expressed relative to an untreated control (UTC). ISIS Number 327917 was observed to stimulate an approximately 2-fold increase in levels of the mir-21 pri-miRNA molecule as detected by real-time RT-PCR.

Thus, it is believed that, in addition to binding the mir-21 mature miRNA and interfering with the RISC-mediated binding of mir-21 to its mRNA target, the oligomeric compound, ISIS 327917, binds to the mir-21 pri-miRNA and/or pre-miRNA molecules and interferes with their processing into the mature mir-21 miRNA, inhibiting expression of the mature mir-21 miRNA in HeLa cells, possibly by interfering with the activity of RNase III-like enzymes such as human Dicer or Drosha. The resultant decrease in levels of mature mir-21 miRNA may trigger a feedback mechanism leading to an upregulation of production of the mir-21 pri-miRNA molecule. Treatment with this oligomeric compound could also stimulate the activity of an RNA-dependent RNA polymerase (RdRP) that amplifies the mir-21 pri-miRNA or pre-miRNA molecules.

In accordance with the present invention, a nested series of uniform 2′-MOE oligomeric compounds were designed and synthesized to target the entire length of the mir-21 pri-miRNA molecule. The region encompassing the mir-21 primary transcript (nucleotides 16571584 to 16571864 of GenBank Accession number NT_(—)010783.14; CTGGGTTTTTTTGGTTTGTTTTTGTTTTTGTTTTTTTATCAAATCCTGCCTGACTGTCTGCTT GTTTTGCCTACCATCGTGACATCTCCATGGCTGTACCACCTTGTCGGGTAGCTTATCAGAC TGATGTTGACTGTTGAATCTCATGGCAACACCAGTCGATGGGCTGTCTGACATTTTGGTA TCTTTCATCTGACCATCCATATCCAATGTTCTCATTTAAACATTACCCAGCATCATTGTTT ATAATCAGAAACTCTGGTCCTTCTGTCTGGTGGCAC) is incorporated herein as SEQ ID NO: 914. Each compound is 20 nucleotides in length, composed of 2′-methoxyethoxy (2′-MOE) nucleotides and phosphorothioate (P═S) internucleoside linkages throughout the compound. All cytidine residues are 5-methylcytidines. The compounds are shown in Table 48. The compounds can be analyzed for their effect on mature miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR, or they can be used in other assays to investigate the role of miRNAs or the function of targets downstream of miRNAs.

TABLE 48 Uniform 2′-MOE PS Compounds targeting the mir-21 pri-miRNA ISIS SEQ ID Number NO Sequence 358765 915 ACAAGCAGACAGTCAGGCAG 358766 916 GGTAGGCAAAACAAGCAGAC 358767 917 GGAGATGTCACGATGGTAGG 358768 918 AGGTGGTACAGCCATGGAGA 358769 919 GATAAGCTACCCGACAAGGT 358770 920 AGTCTGATAAGCTACCCGAC 358771 921 CAACAGTCAACATCAGTCTG 358772 922 GAGATTCAACAGTCAACATC 358773 923 CTGGTGTTGCCATGAGATTC 358774 924 CATCGACTGGTGTTGCCATG 358775 925 ACAGCCCATCGACTGGTGTT 358776 926 TGTCAGACAGCCCATCGACT 358777 927 CCAAAATGTCAGACAGCCCA 358778 928 GATACCAAAATGTCAGACAG 358779 929 GGTCAGATGAAAGATACCAA 358780 930 AACATTGGATATGGATGGTC 358781 931 TAATGTTTAAATGAGAACAT 358782 932 AACAATGATGCTGGGTAATG 358783 933 GAGTTTCTGATTATAAACAA 358784 934 CGACAAGGTGGTACAGCCAT 358785 935 GAAAGATACCAAAATGTCAG

Using the real-time RT-PCR methods, the expression levels of the mir-21 pri-miRNA were compared in HeLa cells treated with this nested series of uniform 2′-MOE oligomeric compounds, targeting and spanning the entire length of the mir-21 pri-miRNA. ISIS Number 358768 (SEQ ID NO: 918) targets a region flanking the predicted 5′ Drosha cleavage site in the mir-21 pri-miRNA. ISIS Number 358777 (SEQ ID NO: 927) targets a region spanning the 3′ Drosha cleavage site in the mir-21 pri-miRNA. ISIS 358779 (SEQ ID NO: 929) targets a region flanking the predicted 3′ Drosha cleavage site in the mir-21 pri-miRNA. Additionally, oligomeric compounds ISIS 327917 (SEQ ID NO: 335), a uniform 2′-MOE compound targeting the mature mir-21 miRNA, and ISIS 345382 (TCAACATCAGTCTGATAAGCTA; SEQ ID NO: 335), a 5-10-7 phosphorothioate 2′-MOE gapmer targeting mir-21, were tested for comparison. Oligomeric compound ISIS 327863 (ACGCTAGCCTAATAGCGAGG; herein incorporated as SEQ ID NO: 936), a phosphorothioate 5-10-52′-MOE gapmer, was used as scrambled control. Using the transfection methods previously described, HeLa cells were treated with 100 nM of each of these oligomeric compounds. Total RNA was isolated by lysing cells in 1 mL TRIZOL™ (Invitrogen) using the manufacturer's recommended protocols. real-time RT-PCR analysis was performed using the primer/probe set specific for the mir-21 pri-miRNA molecule [forward primer=ISIS 339332 (SEQ ID NO.: 911), reverse primer-ISIS 339333 (SEQ ID NO.: 912), and probe=ISIS 339334 (SEQ ID NO.: 913)]. Total RNA was quantified using RiboGreen™ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.), expression levels observed for each target were normalized to 5.8S rRNA, and values were expressed relative to the untreated control (UTC). Effects on expression of the mir-21 pri-miRNA molecule resulting from treatment of HeLa cells with these uniform 2′-MOE oligomeric compounds is expressed as a percentage of RNA levels in untreated control cells. Results of these experiments are shown in Table 49 below:

TABLE 49 Effects of oligomeric compounds on mir-21 pri-miRNA expression % ISIS # SEQ ID NO: target UTC UTC N/A N/A 100 327863 936 N/A 107 gapmer control 327917 335 mature mir-21 249 uniform 2′-MOE 345382 335 mature mir-21 119 5-10-7 2′-MOE gapmer 358765 915 mir-21 pri-miRNA 133 358766 916 mir-21 pri-miRNA 142 358767 917 mir-21 pri-miRNA 248 358768 918 flanking 5′ Drosha cleavage site 987 of mir-21 pri-miRNA 358769 919 mir-21 pri-miRNA 265 358770 920 mir-21 pri-miRNA 250 358771 921 mir-21 pri-miRNA 181 358772 922 mir-21 pri-miRNA 245 358773 923 mir-21 pri-miRNA 148 358774 924 mir-21 pri-miRNA 104 358775 925 mir-21 pri-miRNA 222 358776 926 mir-21 pri-miRNA 367 358777 927 spanning 3′ Drosha cleavage site 536 of mir-21 pri-miRNA 358778 928 mir-21 pri-miRNA 503 358779 929 flanking 3′ Drosha cleavage site 646 of mir-21 pri-miRNA 358780 930 mir-21 pri-miRNA 269 358781 931 mir-21 pri-miRNA 122 358782 932 mir-21 pri-miRNA 155 358783 933 mir-21 pri-miRNA 133 358784 934 mir-21 pri-miRNA 358 358785 935 mir-21 pri-miRNA 257

From these data, it was observed that the uniform 2′-MOE oligomeric compounds ISIS Numbers 327917, 358767, 358768, 358769, 358770, 358772, 358775, 358776, 358777, 358778, 358779, 358780, 358784 and 358785 stimulate an increase in levels of the mir-21 pri-miRNA molecule as detected by real-time RT-PCR. Notably, oligomeric compounds ISIS Numbers 358768 and 358779 which target the regions flanking the predicted 5′ and 3′ Drosha cleavage sites, respectively, and ISIS Number 358777, which targets a region spanning the 3′ Drosha cleavage site in the mir-21 pri-miRNA were observed to stimulate the greatest increases in expression of the mir-21 pri-miRNA. Furthermore, treatment of HeLa cells with increasing concentrations (25, 50, 100, and 200 nM) of ISIS Numbers 358768, 358779, and 327917 was observed to result in a dose-responsive induction of mir-21 pri-miRNA levels. Thus, it is believed that these oligomeric compounds bind to the mir-21 pri-miRNA and/or pre-miRNA molecules and interfere with their processing into the mature mir-21 miRNA, possibly by interfering with the activity of RNase III-like enzymes such as human Dicer and/or Drosha. The resultant decrease in levels of the processed mature mir-21 miRNA may trigger a feedback mechanism leading to an upregulation of production of the mir-21 pri-miRNA molecule. Not mutually exclusive with the processing interference and the feedback mechanisms is the possibility that treatment with oligomeric compounds could stimulate the activity of an RNA-dependent RNA polymerase (RdRP) that amplifies the mir-21 pri-miRNA or pre-miRNA molecules. It is understood that such oligomeric compound-triggered mechanisms may be operating not only upon regulation of mir-21 production and processing, but may also be found to regulate the production and processing of other miRNAs or target nucleic acids.

In one embodiment, the oligomeric compounds ISIS Number 327917 (SEQ ID NO: 335), the phosphorothioate uniform 2′-MOE targeting mature mir-21; ISIS Number 358768 (SEQ ID NO: 918), the uniform 2′-MOE targeting the mir-21 pri-miRNA which stimulated the largest increase in pri-miRNA expression levels by real time quantitative RT-PCR; and ISIS Number 345382 (SEQ ID NO: 335), the 5-10-7 phosphorothioate 2′-MOE gapmer targeting mature mir-21 were selected for dose response studies in HeLa cells using the luciferase reporter system described in Example 27. ISIS Number 342683 (SEQ ID NO: 790), representing the scrambled nucleotide sequence of an unrelated PTP1B antisense oligonucleotide, was used as a negative control. HeLa cells expressing the pGL3-mir-21 sensor plasmid (described in Example 27) were treated with 1.9, 5.5, 16.7, and 50 nM of these oligomeric compounds, to assess the ability of oligomeric compounds to interfere with endogenous mir-21-mediated silencing of the pGL3-mir-21 sensor plasmid. The data are presented in Table 50 as percent untreated control (luciferase plasmid only, not treated with oligomeric compound) luciferase expression, normalized to pRL-CMV levels.

TABLE 50 Effects of oligomeric compounds on mir-21 miRNA-mediated inhibition of luciferase expression % UTC Dose of oligomeric compound Treatment 1.9 nM 5.5 nM 16.7 nM 50 nM 342683 127 171 104 108 negative control 327917 522 1293 2470 4534 358768 103 163 146 118 345382 101 135 117 95

From these data, it was observed that, at all doses, treatment of HeLa cells with ISIS Number 327917, the uniform 2′-MOE oligomeric compound targeting the mature mir-21 miRNA, de-repressed the expression of the luciferase reporter, in a dose-dependent fashion. Thus, ISIS 327917 reversed the silencing effect of the endogenous mir-21 miRNA, possibly by inhibiting the binding of mir-21 to its target site encoded by the pGL3-mir-21 sensor plasmid.

Example 29 Diseases Associated with miRNA-Containing Loci

Using the public databases Online Mendelian Inheritance in Man (OMIM) (accessible through the Internet at, for example, ftp.ncbi.nih.gov/repository/OMIM/) and LocusLink (accessible at, for example, ftp.ncbi.nlm.nih.gov/refseq/LocusLink/), a bioinformatic analysis was performed which allowed the prediction of miRNAs associated with several human diseases. First, miRNAs encoded within genes having LocusLink identification numbers were identified, and these were compared to tables (for example, “mim2loc,” which connects LocusLink identification numbers with OMIM identification numbers, as well as “genemap,” “genemap.key,” “mim-title,” and “morbidmap” tables) for the construction of a new database called “db 1.mdb” linking miRNAs to LocusLink and OMIM identification numbers and linking these to human diseases.

It was observed that, beginning with 95 pri-miRNAs, a subset of 49 had OMIM identification numbers, 48 of which were linked to OMIM names. Six of these miRNAs were associated with specific diseased patients (some in each category were duplicates). Thus, the majority of miRNAs with OMIM identification numbers are not directly linked to observed diseases, but are likely to be important in pathways (such as cholesterol homeostasis) associated with diseases. Tables 51 and 52 summarize information retrieved from these studies.

TABLE 51 miRNA genes associated with specific diseases OMIM ID: locus containing miRNA Disease association: 120150 collagen, type I, alpha 1/ Osteogenesis imperfecta, hypothetical miRNA-144 type I, 166200 114131 calcitonin receptor containing Osteoporosis, hypothetical miRNA 30 postmenopausal susceptibility, 166710 605317 forkhead box P2/ Speech-language disorder- hypothetical miRNA 169 1, 602081 600700 LIM domain-containing preferred Lipoma; Leukemia, translocation partner in lipoma myeloid containing miR-28 160710 myosin, heavy polypeptide 6, Cardiomyopathy, familial cardiac muscle, alpha hypertrophic, 192600 (cardiomyopathy, hypertrophic 1) containing miR-208 606157 hypothetical protein FLJ11729 Neurodegeneration, containing mir-103-2 pantothenate kinase- associated, 234200

The previous table shows miRNAs associated with an OMIM record that were also associated with diseased patients.

The following table, Table 52, describes diseases or disease-related phenotypes found to be associated with genetic loci associated with a miRNA.

TABLE 52 miRNAs associated with disease phenotypes OMIM ID: Locus containing miRNA Disease association: 114131 calcitonin receptor containing Osteoporosis, hypothetical miRNA-30 postmenopausal, susceptibility, 166710 120150 collagen, type I, alpha 1/ Osteogenesis imperfecta, hypothetical miRNA-144 type I, 166200 138247 glutamate receptor, ionotropic, cerebellar long-term AMPA 2/hypothetical miRNA-171 depression 160710 myosin, heavy polypeptide 6, Cardiomyopathy, familial cardiac muscle, alpha hypertrophic, 192600 (cardiomyopathy, hypertrophic 1) containing miR-208 184756 sterol regulatory element-binding Emery-Dreifuss muscular protein-1/mir-33b dystrophy, 310300; dilated cardiomyopathy (CMD1A), 115200; familial partial lipodystrophy (FPLD), 151660 300093 gamma-aminobutyric acid (GABA) A early-onset parkinsonism, or receptor, epsilon Waisman syndrome, 311510; and MRX3 X-linked mental retardation, 309541 305660 gamma-aminobutyric acid (GABA) A manic depressive illness, receptor, alpha 3 containing miR- colorblindness, and G6PD 105 (Mourelatos) and miR-105-2 305915 glutamate receptor, ionotrophic, complex bipolar disorder; AMPA 3/hypothetical miRNA-033 drug addiction 600150 potassium large conductance cardiovascular disease calcium-activated channel, subfamily M, alpha member 1 containing hypothetical miRNA-172 600395 glypican 1 containing miR-149 angiogenesis 600481 Sterol regulatory element binding LDL and cholesterol transcription factor 2 containing homeostasis mir-33a 600592 Minichromosome maintenance increased chromosomal loss, deficient (S. cerevisiae) 7 DNA replication and containing miR-93 (Mourelatos) recombination and miR-25 and miR-94 600700 LIM domain-containing preferred Lipoma; Leukemia, myeloid translocation partner in lipoma containing miR-28 600758 Focal adhesion kinase, p125/ oncogenesis mir-151 601009 tight junction protein 1 (zona peptic ulcer disease and occludens 1)/hypothetical miRNA- gastric carcinoma 183 601029 mesoderm specific transcript intrauterine and postnatal (mouse) homolog containing mir- growth retardation 240* (Kosik) 601698 protein tyrosine phosphatase, insulin-dependent diabetes receptor type, N polypeptide 2 mellitus (IDDM) containing mir-153-2 601773 protein tyrosine phosphatase, insulin-dependent diabetes receptor type, N containing mir- mellitus (IDDM), 222100 153-1 603576 melastatin 1 containing mir-211 metastatic human melanoma 603634 ribosomal protein L5/ colorectal cancers hypothetical miRNA 168-2 603745 slit (Drosophila) homolog 3 congenital diaphragmatic containing mir-218-2 hernia 603746 slit (Drosophila) homolog 2 retinal ganglion cell axon containing mir-218-1 guidance 603803 dachshund (Drosophila) homolog cell proliferation during containing hypothetical miRNA-083 mammalian retinogenesis and pituitary development 605317 forkhead box P2/hypothetical autism & speech-language miRNA 169 disorder-1, 602081 605547 follistatin-like 1 containing systemic rheumatic diseases mir-198 605575 SMC4 (structural maintenance of cell proliferation chromosomes 4, yeast)-like 1 containing mir-16-3 and mir-15b 605766 deleted in lymphocytic leukemia, B-cell chronic lymphocytic 2 containing mir-16-1 and mir- leukemia 15a-1 606157 hypothetical protein FLJ11729 Neurodegeneration, containing mir-103-2 pantothenate kinase- associated, 234200 (3); 606160 pantothenate kinase containing mir-107 pantothenate kinase- associated neurodegeneration 606161 hypothetical protein FLJ12899 pantothenate kinase- containing mir-103-1 associated neurodegeneration

From these data, it was observed that several miRNAs are predicted to be associated with human disease states. For example, several studies of autistic disorder have demonstrated linkage to a similar region of 7q (the AUTS1 locus), leading to the proposal that a single genetic factor on 7q31 contributes to both autism and language disorders, and it has been reported that the FOXP2 gene, located on human 7q31, encoding a transcription factor containing a polyglutamine tract and a forkhead domain, is mutated in a severe monogenic form of speech and language impairment, segregating within a single large pedigree, and is also disrupted by a translocation. In one recent study, association and mutation screening analysis of the FOXP2 gene was performed to assess the impact of this gene on complex language impairments and autism, and it was concluded that coding-region variants in FOXP2 do not underlie the AUTS1 linkage and that the gene is unlikely to play a role in autism or more common forms of language impairment (Newbury, et al., Am. J. Hum. Genet. 2002, 70, 1318-27). However, hypothetical mir-169 is also encoded by this same genetic locus, and it is possible that mutations affecting the hypothetical mir-169 miRNA could underlie the AUTS1 linkage and play a role in language impairment. To this end, oligomeric compounds targeting or mimicking the mir-169 miRNA may prove useful in the study, diagnosis, treatment or amelioration of this disease.

Example 30 Effects of Oligomeric Compounds Targeting miRNAs on Insulin Signaling and Hallmark Gene Expression in HepG2 Cells

Additional oligomeric compounds were screened in the assays described in Example 18. As stated above, insulin inhibits the expression of IGFBP-1, PEPCK-c and follistatin mRNAs.

Protocols for treatment of HepG2 cells and transfection of oligomeric compounds are as described in Example 18. Also as described in Example 18, forty-four hours post-transfection, the cells in the transfected wells were treated with either no insulin (“basal” Experiment 3 (below), for identification of insulin-mimetic compounds) or with 1 nM insulin (“insulin treated” Experiment 4 (below), for identification of insulin sensitizers) for four hours. At the same time, in both plates, cells in some of the un-transfected control wells are treated with 100 nM insulin to determine maximal insulin response. At the end of the insulin or no-insulin treatment (forty-eight hours post-transfection), total RNA is isolated from both the basal and insulin treated (1 nM) 96-well plates, and the amount of total RNA from each sample is determined using a Ribogreen assay (Molecular Probes, Eugene, Oreg.). Real-time PCR is performed on all the total RNA samples using primer/probe sets for three insulin responsive genes: PEPCK-c, IGFBP-1 and follistatin. Expression levels for each gene are normalized to total RNA, and values±standard deviation are expressed relative to the transfectant only and negative control oligonucleotides. The compound ISIS Number 186515 (AGGTAGCTTTGATTATGTAA; SEQ ID NO: 939) is targeted to IGFBP-1 and is a phosphorothioate 5-10-5 MOE gapmer where all cytosines are 5-methylcytosines, as is used as a transfection control. The oligomeric compound ISIS Number 340341 (TAGCTTATCAGACTGATGTTGA; SEQ ID NO: 236) is a uniform 2′-MOE phosphorothioate compound targeted to mir-104 (Mourelatos), ISIS 340362 (GACTGTTGAATCTCATGGCA; SEQ ID NO: 937) is a 5-10-5 gapmer compound also targeted to mir-104 (Mourelatos), and ISIS Number 341813 (AGACACGTGCACTGTAGA; SEQ ID NO: 938) is a uniform 2′-MOE phosphorothioate compound targeted to mir-139. Results of these experiments are shown in Tables 53 and 54.

TABLE 53 Experiment 3: Effects of oligomeric compounds targeting miRNAs on insulin-repressed gene expression in HepG2 cells SEQ ID IGFBP-1 PEPCK-c Follistatin ISIS NO: NO Pri-miRNA (% UTC) (% UTC) (% UTC) UTC N/A N/A 100 100 100  29848 737 N/A 104 100 90 n-mer 186515 939 IGFBP-1 193 70 67 328384 493 hypothetical 139 142 110 miRNA-039 328677 586 hypothetical 208 145 130 miRNA-120 328685 594 mir-219 157 219 100 328691 600 mir-145 105 108 93 328759 668 mir-216 356 98 266 328761 670 hypothetical 118 48 91 miRNA-138 328765 674 mir-215 88 93 87 328773 682 mir-15a-2 148 138 131 328779 688 hypothetical 135 123 109 mir-177 340341 236 mir-104 110 129 94 (Mourelatos) 340362 937 mir-104 157 168 123 (Mourelatos) 341813 938 mir-139 137 121 100

Under “basal” conditions (without insulin), treatments of HepG2 cells with oligomeric compounds of the present invention resulting in decreased mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that the oligomeric compounds have an insulin mimetic effect. Treatments with oligomeric compounds of the present invention resulting in an increase in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds inhibit or counteract the normal insulin repression of mRNA expression of these genes.

From these data, it is evident that the oligomeric compound ISIS Number 328761 targeting hypothetical mir-138, for example, results in a 52% decrease in PEPCK-c mRNA, a marker widely considered to be insulin-responsive. Thus, this oligomeric compound may be useful as a pharmaceutical agent with insulin mimetic properties in the treatment, amelioration, or prevention of diabetes or other metabolic diseases.

TABLE 54 Experiment 4: Effects of oligomeric compounds targeting miRNAs on insulin-sensitization of gene expression in HepG2 cells SEQ IGFBP-1 PEPCK-c Follistatin ISIS NO: ID NO Pri-miRNA (% UTC) (% UTC) (% UTC) UTC(1 nm N/A N/A 100 100 100 insulin)  29848 737 N/A 92 90 95 n-mer 186515 939 IGFBP-1 105 40 39 328384 493 hypothetical 102 114 121 miRNA-039 328677 586 hypothetical 159 117 118 miRNA-120 328685 594 mir-219 143 184 157 328691 600 mir-145 101 97 104 328759 668 mir-216 212 92 224 328761 670 hypothetical 93 55 98 miRNA-138 328765 674 mir-215 94 73 97 328773 682 mir-15a-2 136 93 148 328779 688 hypothetical 128 78 119 mir-177 340341 236 mir-104 113 115 120 (Mourelatos) 340362 937 mir-104 129 104 119 (Mourelatos) 341813 938 mir-139 117 88 102

In HepG2 cells treated with 1 nM insulin, treatments with oligomeric compounds of the present invention resulting in a decrease in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds have an insulin sensitization effect. Treatments with oligomeric compounds of the present invention resulting in an increase in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds inhibit or counteract the normal insulin response of repression of mRNA expression of these genes.

From these data, it is evident that the oligomeric compounds, ISIS Number 328761 targeting hypothetical mir-138 and ISIS Number 328765 targeting mir-215, for example, were observed to result in a 45% and a 27% reduction, respectively, of PEPCK-c mRNA expression, widely considered to be a marker of insulin-responsiveness. Furthermore, mRNA levels of the IGFBP-1 and follistatin genes were also reduced. Thus, these oligomeric compounds may be useful as pharmaceutical agents with insulin-sensitizing properties in the treatment, amelioration, or prevention of diabetes or other metabolic diseases.

Example 31 Adipocyte Assay of Oligomeric Compounds

The effect of several oligomeric compounds of the present invention targeting miRNA target nucleic acids on the expression of markers of cellular differentiation was examined in differentiating adipocytes.

As described in Example 13, some genes known to be upregulated during adipocyte differentiation include HSL, aP2, Glut4 and PPARγ. These genes play important roles in the uptake of glucose and the metabolism and utilization of fats. An increase in triglyceride content is another well-established marker for adipocyte differentiation.

For assaying adipocyte differentiation, expression of the four hallmark genes, HSL, aP2, Glut4, and PPARγ, as well as triglyceride (TG) accumulation were measured as previously described in adipocytes transfected with uniform 2′-MOE or chimeric gapmer phosphorothioate (PS) oligomeric compounds. Triglyceride levels as well as mRNA levels for each of the four adipocyte differentiation hallmark genes are expressed as a percentage of untreated control (UTC) levels. Results are shown in Table 55.

TABLE 55 Effects of oligomeric compounds targeting miRNAs on expression of adipocyte differentiation markers ISIS Number SEQ ID NO TG HSL AP2 Glut4 PPAR gamma UTC N/A 100 100 100 100 100 ISIS-29848 737 89 84 89 96 100 n-mer 327877 295 109 82 77 119 85 327888 306 132 134 102 84 103 327904 322 56 42 65 40 54 327909 327 132 130 88 132 96 327927 345 125 120 114 120 108 327928 346 45 52 77 39 57 327933 351 127 132 82 127 100 327937 355 81 77 76 63 92 327951 369 76 100 91 81 84 327953 371 94 94 92 112 90 327956 374 80 90 102 69 91 327960 378 47 52 52 34 76 328093 395 59 89 97 73 99 328112 414 92 89 73 97 79 328114 416 110 134 123 116 106 328132 434 120 89 81 67 94 328340 449 76 130 85 112 110 328362 471 73 83 59 80 78 328400 509 60 40 34 18 67 328417 526 83 98 87 68 94 328434 543 91 96 85 83 79 328651 560 93 109 84 78 106 328677 586 34 68 61 44 89 328685 594 50 100 73 69 91 328691 600 130 156 166 144 105 328759 668 87 105 108 66 95

For these data, values for triglyceride accumulation above 100 are considered to indicate that the compound has the ability to stimulate triglyceride accumulation, whereas values at or below 100 indicate that the compound inhibits triglyceride accumulation. With respect to leptin secretion, values above 100 are considered to indicate that the compound has the ability to stimulate secretion of the leptin hormone, and values at or below 100 indicate that the compound has the ability to inhibit secretion of leptin. With respect to the four adipocyte differentiation hallmark genes, values above 100 are considered to indicate induction of cell differentiation, whereas values at or below 100 indicate that the compound inhibits differentiation.

Several compounds were found to have remarkable effects. For example, the oligomeric compounds ISIS Number 327904 (SEQ ID NO: 322), targeted to mir-181a-1, ISIS Number 327928 (SEQ ID NO: 346), targeted to mir-29a, ISIS Number 327960 (SEQ ID NO: 378), targeted to mir-215, ISIS Number 328400 (SEQ ID NO: 509), targeted to mir-196-2, and ISIS Number 328677 (SEQ ID NO: 586), targeted to hypothetical miRNA-120 were shown to reduce the expression levels of all five markers of adipocyte differentiation, indicating that these oligomeric compounds have the ability to block adipocyte differentiation. Therefore, these oligomeric compounds may be useful as therapeutic agents with applications in the treatment, attenuation or prevention of obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases as well as in the maintenance of the pluripotent phenotype of stem or precursor cells.

The oligomeric compounds ISIS Number 328691 (SEQ ID NO: 600) targeted to mir-145, ISIS Number 328114 (SEQ ID NO: 416) targeted to hypothetical miRNA-138, and ISIS Number 327927 (SEQ ID NO: 345) targeted to mir-15b are examples of compounds which exhibit an increase in all five markers of adipocyte differentiation. Additionally, the oligomeric compound ISIS Number 327909 (SEQ ID NO: 327) targeted to mir-196-2 exhibited an increase in three of the five markers of adipocyte differentiation. Thus, these oligomeric compounds may be useful as pharmaceutical agents in the treatment of diseases in which the induction of adipocyte differentiation is desirable, such as anorexia, or for conditions or injuries in which the induction of cellular differentiation is desirable, such as Alzheimers disease or central nervous system injury, in which regeneration of neural tissue would be beneficial. Furthermore, these oligomeric compounds may be useful in the treatment, attenuation or prevention of diseases in which it is desirable to induce cellular differentiation and/or quiescence, for example in the treatment of hyperproliferative disorders such as cancer.

Example 32 Effects of Oligomeric Compounds on Endothelial Tube Formation Assay

Angiogenesis is the growth of new blood vessels (veins and arteries) by endothelial cells. This process is important in the development of a number of human diseases, and is believed to be particularly important in regulating the growth of solid tumors. Without new vessel formation it is believed that tumors will not grow beyond a few millimeters in size. In addition to their use as anti-cancer agents, inhibitors of angiogenesis have potential for the treatment of diabetic retinopathy, cardiovascular disease, rheumatoid arthritis and psoriasis (Carmeliet and Jain, Nature, 2000, 407, 249-257; Freedman and Isner, J. Mol. Cell. Cardiol, 2001, 33, 379-393; Jackson et al., Faseb J., 1997, 11, 457-465; Saaristo et al., Oncogene, 2000, 19, 6122-6129; Weber and De Bandt, Joint Bone Spine, 2000, 67, 366-383; Yoshida et al., Histol. Histopathol, 1999, 14, 1287-1294).

Endothelial Tube Formation Assay as a Measure of Angiogenesis:

Angiogenesis is stimulated by numerous factors that promote interaction of endothelial cells with each other and with extracellular matrix molecules, resulting in the formation of capillary tubes. This morphogenic process is necessary for the delivery of oxygen to nearby tissues and plays an essential role in embryonic development, wound healing, and tumor growth (Carmeliet and Jain, Nature, 2000, 407, 249-257). Moreover, this process can be reproduced in a tissue culture assay that evaluates the formation of tube-like structures by endothelial cells. There are several different variations of the assay that use different matrices, such as collagen I (Kanayasu et al., Lipids, 1991, 26, 271-276), Matrigel (Yamagishi et al., J. Biol. Chem., 1997, 272, 8723-8730) and fibrin (Bach et al., Exp. Cell Res., 1998, 238, 324-334), as growth substrates for the cells. In this assay, human umbilical vein endothelial cells (HuVECs) are plated on a matrix derived from the Engelbreth-Holm-Swarm mouse tumor, which is very similar to Matrigel (Kleinman et al., Biochemistry, 1986, 25, 312-318; Madri and Pratt, J. Histochem. Cytochem., 1986, 34, 85-91). Untreated HuVECs form tube-like structures when grown on this substrate. Loss of tube formation in vitro has been correlated with the inhibition of angiogenesis in vivo (Carmeliet and Jain, Nature, 2000, 407, 249-257; Zhang et al., Cancer Res., 2002, 62, 2034-2042), which supports the use of in vitro tube formation as an endpoint for angiogenesis.

In one embodiment, primary human umbilical vein endothelial cells (HuVECs) were used to measure the effects of oligomeric compounds targeted to miRNAs on tube formation activity. HuVECs were routinely cultured in EBM (Clonetics Corporation, Walkersville, Md.) supplemented with SingleQuots supplements (Clonetics Corporation, Walkersville, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence and were maintained for up to 15 passages. HuVECs are plated at 3000 cells/well in 96-well plates. One day later, cells are transfected with oligomeric compounds. The tube formation assay is performed using an in vitro Angiogenesis Assay Kit (Chemicon International, Temecula, Calif.).

A scrambled control compound, ISIS 29848 NNNNNNNNNNNNNN; where N is A, T, C or G; herein incorporated as SEQ ID NO: 737) served as a negative control. ISIS 196103 (AGCCCATTGCTGGACATGCA; incorporated herein as SEQ ID NO: 940) targets integrin beta 3 and was used as a positive control to inhibit endothelial tube formation. ISIS 29248 and ISIS 196103 are chimeric 5-10-5 2′-MOE gapmer oligonucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotides. All cytidine residues are 5-methylcytidines. ISIS 342672 (SEQ ID NO: 789) contains 13 mismatches with respect to the mature mir-143 miRNA, and was also used as a negative control. ISIS 342672 is a uniform 2′-MOE phosphorothioate oligomeric compound 22 nucleotides in length. All cytidine residues are 5-methylcytidines.

Oligomeric compound was mixed with LIPOFECTIN™ (Invitrogen Life Technologies, Carlsbad, Calif.) in OPTI-MEM™ (Invitrogen Life Technologies, Carlsbad, Calif.) to achieve a final concentration of 75 nM of oligomeric compound and 2.25 μg/mL LIPOFECTIN™. Before adding to cells, the oligomeric compound, LIPOFECTIN™ and OPTI-MEM™ were mixed thoroughly and incubated for 0.5 hrs. Untreated control cells received LIPOFECTIN™ only. The medium was removed from the plates and the plates were tapped on sterile gauze. Each well was washed in 150 μL of phosphate-buffered saline. The wash buffer in each well was replaced with 100 μL of the oligomeric compound/OPTI-MEM™/LIPOFECTIN™ cocktail. Compounds targeted to miRNAs were tested in triplicate, and ISIS 29848 was tested in up to six replicates. The plates were incubated for 4 hours at 37° C., after which the medium was removed and the plate was tapped on sterile gauze. 100 μL of full growth medium was added to each well. Fifty hours after transfection, cells are transferred to 96-well plates coated with ECMa-trix™ (Chemicon Inter-national). Under these conditions, untreated HuVECs form tube-like structures. After an overnight incubation at 37° C., treated and untreated cells are inspected by light microscopy. Individual wells are assigned discrete scores from 1 to 5 depending on the extent of tube formation. A score of 1 refers to a well with no tube formation while a score of 5 is given to wells where all cells are forming an extensive tubular network. Results are expressed as a percentage of the level of the tube formation observed in cultures not treated with oligonucleotide, and are shown in Tables 56-59.

TABLE 56 Effect of compounds targeting miRNAs on Tube Formation Activity in HuVECs SEQ ID % Activity Relative ISIS NO: NO: Pri-miRNA to UTC UTC N/A N/A 100 196103 940 Integrin beta 3 35.7 positive control 342672 789 N/A 46.4 negative control 327873 291 mir-140 100.0 327875 293 mir-34 71.4 327876 294 mir-29b-1 50.0 327877 295 mir-16-3 78.6 327878 296 mir-203 57.1 327879 297 mir-7-1 71.4 327880 298 mir-10b 57.1 327881 299 mir-128a 50.0 327882 300 mir-153-1 107.1 327883 301 mir-27b 92.9 327884 302 mir-96 78.6 327885 303 mir-17as/mir-91 50.0 327886 304 mir-123/mir-126as 42.9 327887 305 mir-132 57.1 327888 306 mir-108-1 100.0 327889 307 mir-23b 50.0 327890 308 let-7i 92.9 327891 309 mir-212 50.0 327892 310 mir-131-2/mir-9 57.1 327893 311 let-7b 100.0 327894 312 mir-1d 100.0 327895 313 mir-122a 100.0 327896 314 mir-22 64.3 327898 316 mir-142 100.0

From these data, it was observed that ISIS Number 327886 targeted to mir-123/mir126 as suppressed tube formation, indicating that this compound may be useful as an angiogenesis inhibitor and/or anti-tumor agent, with potential therapeutic applications in the treatment of diabetic retinopathy, cardiovascular disease, rheumatoid arthritis, psoriasis, as well as cancer.

TABLE 57 Effect of compounds targeting miRNAs on Tube Formation Activity in HuVECs SEQ ID % Activity ISIS NO: NO: Pri-miRNA Relative to UTC UTC N/A N/A 100 196103 940 Integrin beta 3 24.1 positive control 342672 789 N/A 58.6 negative control 327899 317 mir-183 34.5 327900 318 mir-214 55.2 327901 319 mir-143 48.3 327902 320 mir-192-1 41.4 327903 321 let-7a-3 103.5 327904 322 mir-181a 89.7 327905 323 mir-205 48.3 327906 324 mir-103-1 69.0 327907 325 mir-26a 62.1 327908 326 mir-33a 103.5 327909 327 mir-196-2 96.6 327910 328 mir-107 55.2 327911 329 mir-106 75.9 327913 331 mir-29c 69.0 327914 332 mir-130a 82.8 327915 333 mir-218-1 69.0 327916 334 mir-124a-2 96.6 327917 335 mir-21 82.8 327918 336 mir-144 96.6 327919 337 mir-221 103.5 327920 338 mir-222 41.4 327921 339 mir-30d 96.6 327922 340 mir-19b-2 89.7 327923 341 mir-128b 48.3

From these data, it was observed that ISIS Number 327899 targeted to mir-183, ISIS Number 327902 targeted to mir-192-1, and ISIS Number 327920 targeted to mir-222 suppressed tube formation, indicating that these compounds may be useful as an angiogenesis inhibitors and/or anti-tumor agents, with potential therapeutic applications in the treatment of diabetic retinopathy, cardiovascular disease, rheumatoid arthritis, psoriasis, as well as cancer.

TABLE 58 Effect of compounds targeting miRNAs on Tube Formation Activity in HuVECs SEQ ID % Activity Relative ISIS NO: NO: Pri-miRNA to UTC UTC N/A N/A 100 196103 940 Integrin beta 3 29.6 positive control 342672 789 N/A 55.6 negative control 327924 342 mir-129-2 88.9 327925 343 mir-133b 44.4 327926 344 let-7d 96.3 327927 345 mir-15b 59.3 327928 346 mir-29a-1 37.0 327929 347 mir-199b 51.9 327930 348 let-7e 88.9 327931 349 let-7c 103.7 327932 350 mir-204 51.9 327933 351 mir-145 59.3 327934 352 mir-213/mir-181a 51.9 327935 353 mir-20 74.1 327936 354 mir-133a-1 51.9 327937 355 mir-138-2 88.9 327938 356 mir-98 96.3 327939 357 mir-125b-1 66.7 327940 358 mir-199a-2 59.3 327941 359 mir-181b 74.1 327942 360 mir-141 74.1 327943 361 mir-18 81.5 327944 362 mir-220 37.0 327945 363 mir-24-2 59.3 327946 364 mir-211 51.9 327947 365 mir-101-3 81.5

From these data, it was observed that ISIS Number 327925 targeted to mir-133b, ISIS Number 327928 targeted to mir-29a-1, and ISIS Number 327944 targeted to mir-220 suppressed tube formation, indicating that these compounds may be useful as an angiogenesis inhibitors and/or anti-tumor agents, with potential therapeutic applications in the treatment of diabetic retinopathy, cardiovascular disease, rheumatoid arthritis, psoriasis, as well as cancer.

TABLE 59 Effect of compounds targeting miRNAs on Tube Formation Activity in HuVECs SEQ ID % Activity Relative ISIS Number NO: Pri-miRNA to UTC UTC N/A N/A 100 196103 940 Integrin beta 3 26.7 positive control 342672 789 N/A 60.0 negative control 327874 292 mir-30a 46.7 327897 315 mir-92-1 40.0 327901 319 mir-143 100.0 327948 366 mir-30b 33.3 327949 367 mir-10a 66.7 327950 368 mir-19a 73.3 327951 369 mir-15a-1 73.3 327952 370 mir-137 53.3 327953 371 mir-219 53.3 327954 372 mir-148b 53.3 327955 373 mir-130b 46.7 327956 374 mir-216 46.7 327957 375 mir-100-1 66.7 327958 376 mir-187 40.0 327959 377 mir-210 40.0 327960 378 mir-215 53.3 327961 379 mir-223 53.3 327962 380 mir-30c 53.3 327963 381 mir-26b 93.3 327964 382 mir-152 86.7 327965 383 mir-135-1 100.0 327966 384 mir-217 40.0 327967 385 let-7g 93.3 327968 386 mir-33b 93.3

From these data, it was observed that ISIS Number 327948 targeted to mir-30b, ISIS Number 327958 targeted to mir-187, ISIS Number 327959 targeted to mir-210, and ISIS Number 327966 targeted to mir-217 suppressed tube formation, indicating that these compounds may be useful as an angiogenesis inhibitors and/or anti-tumor agents, with potential therapeutic applications in the treatment of diabetic retinopathy, cardiovascular disease, rheumatoid arthritis, psoriasis, as well as cancer.

Example 33 Effect of Oligomeric Compounds on miRNA Target Protein Expression

Several mRNA transcripts have been predicted to be regulated by miRNAs (Lewis et al., Cell, 2003, 115, 787-798). For example, the mRNAs encoded by six genes, 1) inwardly rectifying potassium channel Kir2.2 (GenBank Accession AB074970, SEQ ID NO: 872); 2) synaptotagmin III (GenBank Accession BC028379, SEQ ID NO: 873); 3) mitogen-activated protein kinase 7/extracellular signal-regulated kinase 5 (ERK5) (GenBank Accession NM_(—)139032.1, SEQ ID NO: 861); 4) protein phosphatase 2 (formerly 2A), catalytic subunit, beta isoform (PPP2CB) (GenBank Accession NM_(—)004156.1, SEQ ID NO: 814); 5) glyoxalase I (GenBank Accession NM_(—)006708.1, SEQ ID NO: 821); and 6) LIM domain only 4 (LMO4) (GenBank Accession NM_(—)006769.2, SEQ ID NO: 865), are believed to have mir-143 binding sites within their 3′-UTRs. The latter three genes encode mRNAs that were identified as potential targets of mir-143 by the RACE-PCR experiments described, supra. Thus, the mir-143 miRNA is predicted to regulate some or all of these genes.

When miRNAs have effects on the expression of downstream genes or proteins encoded by these genes, it is advantageous to measure the protein levels of those gene products, and to do this, western blot (immunoblot) analysis is employed. Immunoblot analysis is carried out using standard methods. Briefly, preadipocytes and differentiating adipocytes were cultured as described previously, and differentiating adipocytes are sampled at several timepoints after stimulation of differentiation. Cells were treated with 250 nM oligomeric compounds and harvested 16-20 h after oligomeric compound treatment. Cells were washed, lysed in RIPA buffer with protease inhibitor cocktail (Roche Diagnostics Corporation, Indianapolis, Ind.), suspended in Laemmli buffer (20 ul/well), boiled for 5 minutes and loaded onto either an 8% SDS-PAGE or a 4-20% gradient SDS-PAGE gel. Gels are run for approximately 1.5 hours at 150 V, and transferred to PVDF membrane for western blotting. Appropriate primary antibody directed to a target is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Because expression levels of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein remain constant, an antibody recognizing the GAPDH protein (Abcam, Cambridge, Mass.) can be used in a re-probing of the membrane to verify equal protein loading. It is also understood that antisense oligomeric compounds specifically targeting and known to inhibit the expression of the mRNA and protein endproducts of the gene of interest can be used as controls in these experiments. Bands are visualized and quantitated using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.) or the ChemiDoc™ system (Bio-Rad Laboratories, Inc., Hercules, Calif.). Thus, the effects of treatment of many cell types (including, but not limited to, preadipocytes, differentiating adipocytes, HeLa, T-24 and A549 cells) with oligomeric compounds of the present invention on the levels of gene expression products can be assessed. It is understood that one of ordinary skill in the art can use immunoblot analysis to examine the expression of any protein predicted to be the downstream expression product of a target of a miRNA. Similarly, using methods described above, real-time RT-PCR methods can also be used to examine the mRNA expression levels of any of these predicted targets of the mir-143 miRNA. More specifically, immunoblot analysis and/or real-time RT-PCR methods can be used to examine the effects of treatment with oligomeric compounds on the protein or mRNA levels, respectively, produced by the Kir2.2, synaptotagmin III, ERK5, PPP2CB, glyoxalase I, and/or LMO4 genes in a variety of cell types.

In one embodiment of the invention, immunoblot analysis was used to assess the effects of the oligomeric compound, ISIS Number 327901 (SEQ ID NO: 319) targeting mir-143, on expression levels of the PPP2CB protein in differentiating adipocytes. It was observed that, upon treatment with ISIS 327901, PPP2CB protein levels were higher in differentiating adipocytes both 7- and 10-days post-differentiation than in pre-adipocytes or in untreated differentiating adipocytes from the same timepoints. Thus, mir-143 appears to negatively regulate the expression of the PPP2CB gene, presumably by inhibiting translation of the PPP2CB mRNA into protein, and upon treatment with the oligomeric compound ISIS 327901, this inhibition of PPP2CB protein expression was relieved.

In one embodiment of the invention, immunoblot analysis was used to assess the effects of the oligomeric compound, ISIS Number 327901 (SEQ ID NO: 319) targeting mir-143, on expression levels of the ERK5 protein in differentiating adipocytes. It was observed that, upon treatment of cells with ISIS 327901, ERK5 protein levels were approximately 2-2.5-fold higher in differentiating adipocytes both 7- and 10-days post-differentiation than in pre-adipocytes or in untreated differentiating adipocytes from the same timepoints. Thus, mir-143 appears to negatively regulate the expression of the ERK5 gene presumably by inhibiting translation of the ERK5 mRNA into protein, either directly (by mir-143 binding an ERK5 cis-regulatory sequence) or indirectly (by mir-143 regulating another target gene that regulates ERK5); upon treatment with the oligomeric compound ISIS 327901, this mir-143-dependent inhibition of ERK5 expression was relieved. It is known that ERK5 promotes cell growth and proliferation in response to tyrosine kinase signaling. In light of the involvement of mir-143 in adipocyte differentiation disclosed in several examples in the present invention, as well as the role of mir-143 in regulating ERK5, it is predicted that ERK5 and mir-143 are together involved regulating the balance between cellular proliferation and differentiation.

It is understood that the oligomeric compounds of the present invention, including miRNA mimics, can also be tested for their effects on the expression of the protein endproducts of targets of miRNAs. For example, an oligomeric compound such as a mir-143 mimic can be used to treat differentiating adipocytes, and is predicted to result in a reduction of Kir2.2, synaptotagmin III, ERK5, PPP2CB, glyoxalase I, and/or LMO4 protein expression levels.

The phosphatase and tensin homolog (mutated in multiple advanced cancers 1) (PTEN) tumor suppressor mRNA (GenBank Accession NM_(—)000314, incorporated herein as SEQ ID NO: 941) has been predicted to be a potential target of the mir-19a miRNA (Lewis et al., Cell, 2003, 115, 787-798). Oligomeric compounds that target or mimic the mir-19a miRNA or mir-19a pri-miRNA can be used to treat cells and, using the methods described above, the effects of these oligomeric compounds on the expression of the PTEN protein and mRNA levels can be assessed. It is predicted that the mir-19a miRNA, or an oligomeric compound acting as a mir-19a mimic, would inhibit expression of the PTEN tumor suppressor mRNA and protein, and that treatment with oligomeric compounds targeting mir-19a would reverse this inhibition. It is also understood that other antisense oligomeric compounds specifically targeting and known to inhibit the expression of the mRNA and protein endproducts of the gene interest can be used as controls in these experiments.

Example 34 Additional Oligomeric Compounds Targeting miRNAs

In accordance with the present invention, oligomeric compounds were designed and synthesized to target or mimic one or more miRNA genes or gene products. Pri-miRNAs, pre-miRNAs and mature miRNAs represent target nucleic acids to which the oligomeric compounds of the present invention were designed and synthesized. Oligomeric compounds of the present invention can also be designed and synthesized to mimic the pri-miRNA, pre-miRNA or mature miRNA structure while incorporating certain chemical modifications that alter one or more properties of the mimic, thus creating a construct with superior properties as compared to the endogenous precursor or mature miRNA.

In accordance with the present invention, oligomeric compounds were designed to target or mimic one or more human, mouse, rat, or Drosophila pri-miRNAs, pre-miRNAs or mature miRNAs.

A list of human pri-miRNAs and the mature miRNAs predicted to derive from them is shown in Table 60. “Pri-miRNA name” indicates the gene name for each of the pri-miRNAs. Also given in table 60 are the name and sequence of the mature miRNA derived from the pri-miRNA. Mature miRNA sequences from pri-miRNA precursors have been proposed by several groups; consequently, for a given pri-miRNA sequence, several miRNAs may be disclosed and given unique names, and thus a given pri-miRNA sequence may occur repeatedly in the table. The sequences are written in the 5′ to 3′ direction and are represented in the DNA form. It is understood that a person having ordinary skill in the art would be able to convert the sequence of the targets to their RNA form by simply replacing the thymidine (T) with uracil (U) in the sequence.

TABLE 60 Human pri-miRNA sequences and the corresponding mature miRNAs SEQ SEQ ID ID Pri-miRNA name NO Mature miRNA name Mature miRNA sequence NO mir-27b 17 mir-27b TTCACAGTGGCTAAGTTCTG 202 mir-27b 17 miR-27* (Michael TTCACAGTGGCTAAGTTCTGC 1059 et al) mir-23b 23 mir-23b ATCACATTGCCAGGGATTACCAC 208 glutamate 36 hypothetical TGTTATAGTATTCCACCTACC 1060 receptor, miRNA-033 ionotrophic, AMPA 3/hypothetical miRNA-033 LOC 114614 74 hypothetical TGCTAATCGTGATAGGGGTTT 1061 containing miR- miRNA-071 155/hypothetical miRNA-071 LOC 114614 74 mir-155 (RFAM) TTAATGCTAATCGTGATAGGGG 1062 containing miR- 155/hypothetical miRNA-071 collagen, type I, 147 hypothetical AGACATGTTCAGCTTTGTGGA 1063 alpha 1/ miRNA-144 hypothetical miRNA-144 sterol regulatory 168 mir-33b GTGCATTGCTGTTGCATTG 286 element-binding protein-1/mir-33b tight junction 186 hypothetical AGCCTGTGGAGCTGCGCTTAC 1064 protein 1 (zona miRNA-183 occludens 1)/ hypothetical miRNA-183 mir-140 4 mir-140 AGTGGTTTTACCCTATGGTAG 192 mir-140 4 miR-140-as TACCACAGGGTAGAACCACGGA 1065 mir-140 4 mir-239* (Kosik) TACCACAGGGTAGAACCACGGACA 1066 mir-34 6 mir-34 TGGCAGTGTCTTAGCTGGTTGT 194 mir-34 6 miR-172 (RFAM-M. mu.) TGGCAGTGTCTTAGCTGGTTGTT 1067 mir-203 10 mir-203 GTGAAATGTTTAGGACCACTAG 197 mir-203 10 miR-203 (RFAM-M. mu.) TGAAATGTTTAGGACCACTAG 1068 mir-203 10 miR-203 (Tuschl) TGAAATGTTTAGGACCACTAGA 1069 mir-7_1/mir-7_1* 11 mir-7_1*_Ruvkun CAACAAATCACAGTCTGCCATA 1070 mir-7_1/mir-7_1* 11 mir-7 TGGAAGACTAGTGATTTTGTT 198 mir-10b 12 miR-10b (Tuschl) CCCTGTAGAACCGAATTTGTGT 1071 mir-10b 12 mir-10b TACCCTGTAGAACCGAATTTGT 199 mir-10b 12 miR-10b (Michael TACCCTGTAGAACCGAATTTGTG 1072 et al) mir-128a 13 mir-128 (Kosik) TCACAGTGAACCGGTCTCTTT 1073 mir-128a 13 mir-128a TCACAGTGAACCGGTCTCTTTT 200 mir-153_1 14 mir-153 TTGCATAGTCACAAAAGTGA 201 mir-153_2 15 mir-153 TTGCATAGTCACAAAAGTGA 201 hypothetical miR- 16 hypothetical TATCAAACATATTCCTACAGT 1074 13/miR-190 miRNA-013 hypothetical miR- 16 miR-190 TGATATGTTTGATATATTAGGT 1075 13/miR-190 mir-123/mir-126 20 mir-123/mir-126as CATTATTACTTTTGGTACGCG 205 mir-123/mir-126 20 mir-126 TCGTACCGTGAGTAATAATGC 1076 mir-132 21 miR-132 (RFAM- TAACAGTCTACAGCCATGGTCG 1077 Human) mir-132 21 mir-132 TAACAGTCTACAGCCATGGTCGC 206 mir-108_1 22 mir-108 ATAAGGATTTTTAGGGGCATT 207 let-7i 24 let-7i TGAGGTAGTAGTTTGTGCT 209 let-7i 24 let-7i_Ruvkun TGAGGTAGTAGTTTGTGCTGTT 1078 mir-212 25 mir-212 TAACAGTCTCCAGTCACGGCC 210 hypothetical miRNA 26 hypothetical TGGGCAAGAGGACTTTTTAAT 1079 023 miRNA-023 mir-131_2/mir-9 27 mir-131 TAAAGCTAGATAACCGAAAGT 211 mir-131_2/mir-9 27 mir-131_Ruvkun TAAAGCTAGATAACCGAAAGTA 1080 mir-131_2/mir-9 27 miR-9 TCTTTGGTTATCTAGCTGTATGA 1081 let-7b 28 let-7b TGAGGTAGTAGGTTGTGTGGTT 212 let-7b 28 let-7b_Ruvkun TGAGGTAGTAGGTTGTGTGGTTT 1082 mir-1d_1 29 miR-1 (RFAM) TGGAATGTAAAGAAGTATGTA 1083 mir-1d_1 29 mir-1d TGGAATGTAAAGAAGTATGTAT 213 mir-122a 30 miR-122a,b TGGAGTGTGACAATGGTGTTTG 1084 (Tuschl) mir-122a 30 mir-122a TGGAGTGTGACAATGGTGTTTGT 214 mir-22 31 mir-22 AAGCTGCCAGTTGAAGAACTGT 215 hypothetical miRNA 33 hypothetical TGACATCACATATACGGCAGC 1085 30 miRNA-030 mir-142 34 mir-142 CATAAAGTAGAAAGCACTAC 217 mir-142 34 miR-142-as TGTAGTGTTTCCTACTTTATGG 1086 mir-142 34 miR-142as TGTAGTGTTTCCTACTTTATGGA 1087 (Michael et al) mir-183 35 mir-183 TATGGCACTGGTAGAATTCACTG 218 mir-214 37 mir-214 ACAGCAGGCACAGACAGGCAG 219 mir-143 38 miR-143 (Michael TGAGATGAAGCACTGTAGCTC 1088 et al) mir-143 38 mir-143 TGAGATGAAGCACTGTAGCTCA 220 mir-192_1 39 miR-192 (Tuschl) CTGACCTATGAATTGACA 1089 mir-192_1 39 mir-192 CTGACCTATGAATTGACAGCC 221 mir-192_1 39 miR-192 (Michael TGACCTATGAATTGACAGCCAG 1090 et al) hypothetical miRNA 42 hypothetical TAAGACTTGCAGTGATGTTTA 1091 039 miRNA-039 hypothetical miRNA 43 hypothetical TGTCAACAAAACTGCTTACAA 1092 040 miRNA-040 hypothetical miRNA 44 hypothetical TACCAGTTGTTTTCTCTGTGA 1093 041 miRNA-041 let-7a_3 45 let-7a TGAGGTAGTAGGTTGTATAGTT 222 hypothetical miRNA 46 hypothetical TGACAGGAAATCTTTGAGAGG 1094 043 miRNA-043 hypothetical miRNA 47 hypothetical TTCCACTCTGTTTATCTGACA 1095 044 miRNA-044 mir-181a_1 48 mir-178 (Kosik) AACATTCAACGCTGTCGGTGAG 1096 mir-181a_1 48 mir-181a AACATTCAACGCTGTCGGTGAGT 223 let-7a_1 49 let-7a TGAGGTAGTAGGTTGTATAGTT 222 mir-205 50 mir-205 TCCTTCATTCCACCGGAGTCTG 224 mir-33a 53 mir-33a GTGCATTGTAGTTGCATTG 227 mir-196_2 54 miR-196 (Tuschl) TAGGTAGTTTCATGTTGTTGG 1097 mir-196_2 54 mir-196 TAGGTAGTTTCATGTTGTTGGG 228 let-7f_1 57 let-7f (Michael TGAGGTAGTAGATTGTATAGT 1098 et al) let-7f_1 57 let-7f TGAGGTAGTAGATTGTATAGTT 231 hypothetical miRNA 58 hypothetical TTGCATGCCCTATTGATTCTC 1099 055 miRNA-055 mir-29c 59 mir-29c CTAGCACCATTTGAAATCGGTT 232 mir-29c 59 miR-29c (Tuschl) TAGCACCATTTGAAATCGGTTA 1100 mir-130a 60 mir-130a CAGTGCAATGTTAAAAGGGC 233 mir-130a 60 mir-130 (Kosik) CAGTGCAATGTTAAAAGGGCAT 1101 hypothetical miRNA 61 hypothetical TGTCAGATGCTTAATGTTCTT 1102 058 miRNA-058 mir-218_1 62 mir-218 TTGTGCTTGATCTAACCATGT 234 mir-218_1 62 mir-253* (Kosik) TTGTGCTTGATCTAACCATGTG 1103 mir-124a_2 63 mir-124a (Kosik) TAAGGCACGCGGTGAATGCCA 1104 mir-124a_2 63 mir-124a TTAAGGCACGCGGTGAATGCCA 235 mir-124a_2 63 mir-124a_Ruvkun TTAAGGCACGCGGTGAATGCCAA 1105 mir-144 66 mir-144 TACAGTATAGATGATGTACTAG 237 mir-221 67 mir-221 (RFAM- AGCTACATTGTCTGCTGGGTTT 1106 mmu) mir-221 67 mir-221 AGCTACATTGTCTGCTGGGTTTC 238 mir-222 68 mir-222 (RFAM- AGCTACATCTGGCTACTGGGTCT 1107 mmu) mir-222 68 mir-222 AGCTACATCTGGCTACTGGGTCTC 239 mir-30d 69 mir-30d TGTAAACATCCCCGACTGGAAG 240 mir-30d 69 mir-30d_Ruvkun TGTAAACATCCCCGACTGGAAGCT 1108 mir-128b 71 mir-128 (Kosik) TCACAGTGAACCGGTCTCTTT 1073 mir-128b 71 mir-128b TCACAGTGAACCGGTCTCTTTC 242 mir-219_2 72 mir-219 TGATTGTCCAAACGCAATTCT 271 hypothetical miRNA 73 hypothetical TCACATTTGCCTGCAGAGATT 1109 070 miRNA-070 mir-129_2 76 mir-129as/mir- AAGCCCTTACCCCAAAAAGCAT 1110 258* (Kosik) mir-129_2 76 mir-129 CTTTTTGCGGTCTGGGCTTGC 243 mir-129_2 76 miR-129b (RFAM- CTTTTTGCGGTCTGGGCTTGCT 1111 Human) mir-133b 77 mir-133b TTGGTCCCCTTCAACCAGCTA 244 hypothetical miRNA 78 hypothetical TGGTTAAAATATTAATGGGGC 1112 075 miRNA-075 let-7d 79 let-7d AGAGGTAGTAGGTTGCATAGT 245 let-7d 79 let-7d_Ruvkun AGAGGTAGTAGGTTGCATAGTT 1113 let-7d 79 let-7d* (RFAM-M. mu.) CTATACGACCTGCTGCCTTTCT 1114 mir-15b 80 miR-15b (Michael TAGCAGCACATCATGGTTTAC 1115 et al) mir-15b 80 mir-15b TAGCAGCACATCATGGTTTACA 246 mir-29a 81 mir-29a CTAGCACCATCTGAAATCGGTT 247 mir-29a 81 mir-29a_Ruvkun TAGCACCATCTGAAATCGGTTA 1116 hypothetical miRNA 82 hypothetical TGATATGTTTGATATTGGG 1117 079 miRNA-079 mir-199b 83 mir-199b (human) CCCAGTGTTTAGACTATCTGTTC 248 mir-199b 83 miR-199-as TACAGTAGTCTGCACATTGGTT 1118 mir-129_1 84 mir-129 CTTTTTGCGGTCTGGGCTTGC 243 mir-129_1 84 miR-129b (RFAM- CTTTTTGCGGTCTGGGCTTGCT 1111 Human) let-7e 85 let-7e TGAGGTAGGAGGTTGTATAGT 249 hypothetical miRNA 86 hypothetical TTACATGGGGAAGCTATCATA 1119 083 miRNA-083 let-7c_1 87 let-7c TGAGGTAGTAGGTTGTATGGTT 250 let-7c_1 87 let-7c_Ruvkun TGAGGTAGTAGGTTGTATGGTTT 1120 mir-204 88 mir-204 TTCCCTTTGTCATCCTATGCCT 251 mir-204 88 miR-204 (Tuschl) TTCCCTTTGTCATCCTATGCCTG 1121 mir-145 89 miR-145 (Michael GTCCAGTTTTCCCAGGAATCC 1122 et al) mir-145 89 mir-145 GTCCAGTTTTCCCAGGAATCCCTT 252 mir-124a_1 90 mir-124a (Kosik) TAAGGCACGCGGTGAATGCCA 1104 mir-124a_1 90 mir-124a TTAAGGCACGCGGTGAATGCCA 235 mir-124a_1 90 mir-124a_Ruvkun TTAAGGCACGCGGTGAATGCCAA 1105 DiGeorge syndrome 91 hypothetical TGTGATTTCCAATAATTGAGG 1123 critical region miRNA-088 gene 8/ hypothetical miRNA-088 mir-213/mir- 92 mir-178 (Kosik) AACATTCAACGCTGTCGGTGAG 1096 181a_2 mir-213/mir- 92 mir-181a AACATTCAACGCTGTCGGTGAGT 223 181a_2 mir-213/mir- 92 mir-213 ACCATCGACCGTTGATTGTACC 253 181a_2 hypothetical miRNA 93 hypothetical TAGGCCAAATGGCGCATCAAT 1124 090 miRNA-090 mir-20 94 miR-20* (human) ACTGCATTATGAGCACTTAAA 1125 mir-20 94 miR-20 (RFAM- TAAAGTGCTTATAGTGCAGGTA 1126 Human) mir-20 94 mir-20 TAAAGTGCTTATAGTGCAGGTAG 254 mir-133a_1 95 mir-133a TTGGTCCCCTTCAACCAGCTGT 255 mir-138_2 96 mir-138 AGCTGGTGTTGTGAATC 256 mir-138_2 96 mir-138_Ruvkun AGCTGGTGTTGTGAATCAGGCCG 1127 mir-196_1 98 miR-196 (Tuschl) TAGGTAGTTTCATGTTGTTGG 1097 mir-196_1 98 mir-196 TAGGTAGTTTCATGTTGTTGGG 228 mir-125b_1 99 mir-125b TCCCTGAGACCCTAACTTGTGA 258 mir-199a_2 100 miR-199-s CCCAGTGTTCAGACTACCTGTT 1128 mir-199a_2 100 mir-199a CCCAGTGTTCAGACTACCTGTTC 259 mir-199a_2 100 miR-199-as TACAGTAGTCTGCACATTGGTT 1118 hypothetical miRNA 102 hypothetical AGGCAGATAGAGAAGTCACAG 1272 099 miRNA-099 mir-181b_1 103 mir-181b AACATTCATTGCTGTCGGTGGGTT 260 hypothetical miRNA 104 hypothetical TGACAGTCAATTAACAAGTTT 1130 101 miRNA-101 mir-141 105 mir-141 AACACTGTCTGGTAAAGATGG 261 mir-131_1/mir-9 106 mir-131 TAAAGCTAGATAACCGAAAGT 211 mir-131_1/mir-9 106 mir-131_Ruvkun TAAAGCTAGATAACCGAAAGTA 1080 mir-131_1/mir-9 106 miR-9 TCTTTGGTTATCTAGCTGTATGA 1081 mir-133a_2 107 mir-133a TTGGTCCCCTTCAACCAGCTGT 255 hypothetical miRNA 108 miR-202 (human) AGAGGTATAGGGCATGGGAAAA 1131 105 hypothetical miRNA 108 hypothetical TTCCTATGCATATACTTCTTT 1132 105 miRNA-105 hypothetical miRNA 110 hypothetical TGACAGTTTATTGGCTTTATC 1133 107 miRNA-107 mir-1d_2 111 miR-1 (RFAM) TGGAATGTAAAGAAGTATGTA 1083 mir-1d_2 111 mir-1d TGGAATGTAAAGAAGTATGTAT 213 mir-1d_2 111 miR-1d (Tuschl) TGGAATGTAAAGAAGTATGTATT 1134 mir-220 113 mir-220 CCACACCGTATCTGACACTTT 263 hypothetical miRNA 114 hypothetical TTCCTCCTCCTCCGACTCGGA 1135 111 miRNA-111 mir-7_3 115 mir-7 TGGAAGACTAGTGATTTTGTT 198 mir-218_2 116 mir-218 TTGTGCTTGATCTAACCATGT 234 mir-218_2 116 mir-253* (Kosik) TTGTGCTTGATCTAACCATGTG 1103 mir-211 120 mir-211 (human) TTCCCTTTGTCATCCTTCGCCT 1136 mir-30b 122 mir-30b TGTAAACATCCTACACTCAGC 266 mir-30b 122 mir-30b_Ruvkun TGTAAACATCCTACACTCAGCT 1137 hypothetical miRNA 123 hypothetical TTACAGCAATCCAGTAATGAT 1138 120 miRNA-120 mir-10a 125 mir-10a (Tuschl) TACCCTGTAGATCCGAATTTGT 1139 mir-10a 125 mir-10a TACCCTGTAGATCCGAATTTGTG 267 let-7f_2 127 let-7f (Michael TGAGGTAGTAGATTGTATAGT 1098 et al) let-7f_2 127 let-7f TGAGGTAGTAGATTGTATAGTT 231 mir-108_2 129 mir-108 ATAAGGATTTTTAGGGGCATT 207 mir-137 130 mir-137 TATTGCTTAAGAATACGCGTAG 270 mir-148b 132 mir-148b TCAGTGCATCACAGAACTTTGT 272 mir-130b 133 mir-130b CAGTGCAATGATGAAAGGGC 273 mir-130b 133 mir-266* (Kosik) CAGTGCAATGATGAAAGGGCAT 1140 let-7a_4 135 let-7a TGAGGTAGTAGGTTGTATAGTT 222 mir-216 136 mir-216 TAATCTCAGCTGGCAACTGTG 274 hypothetical miRNA 140 hypothetical TAAACTGGCTGATAATTTTTG 1141 137 miRNA-137 hypothetical miRNA 141 hypothetical TGCAAGTATGAAAATGAGATT 1142 138 miRNA-138 mir-124a_3 143 mir-124a (Kosik) TAAGGCACGCGGTGAATGCCA 1104 mir-124a_3 143 mir-124a TTAAGGCACGCGGTGAATGCCA 235 mir-124a_3 143 mir-124a_Ruvkun TTAAGGCACGCGGTGAATGCCAA 1105 mir-7_2 144 mir-7 TGGAAGACTAGTGATTTTGTT 198 hypothetical miRNA 145 hypothetical TGACGCTGCTCCCCACCTTCT 1143 142 miRNA-142 hypothetical miRNA 146 hypothetical TGCAATTTGCTTGCAATTTTG 1144 143 miRNA-143 mir-210 148 mir-210 CTGTGCGTGTGACAGCGGCTG 277 mir-215 149 mir-215 ATGACCTATGAATTGACAGAC 278 mir-223 150 mir-223 TGTCAGTTTGTCAAATACCCC 279 mir-131_3/mir-9 151 mir-131 TAAAGCTAGATAACCGAAAGT 211 mir-131_3/mir-9 151 mir-131_Ruvkun TAAAGCTAGATAACCGAAAGTA 1080 mir-131_3/mir-9 151 miR-9 TCTTTGGTTATCTAGCTGTATGA 1081 mir-199a_1 152 miR-199-s CCCAGTGTTCAGACTACCTGTT 1128 mir-199a_1 152 mir-199a CCCAGTGTTCAGACTACCTGTTC 259 mir-199a_1 152 miR-199-as TACAGTAGTCTGCACATTGGTT 1118 mir-30c_1 153 mir-30c TGTAAACATCCTACACTCTCAGC 280 mir-30c_1 153 mir-30c_Ruvkun TGTAAACATCCTACACTCTCAGCT 1129 hypothetical miRNA 156 hypothetical TGCAAGCAGATGCTGATAATA 1145 153 miRNA-153 hypothetical miRNA 157 hypothetical TTAAAGTGGATGTGTGTTATT 1146 154 miRNA-154 mir-26b 158 miR-26b (RFAM- TTCAAGTAATTCAGGATAGGT 1147 Human) mir-26b 158 mir-26b TTCAAGTAATTCAGGATAGGTT 281 hypothetical miRNA 159 hypothetical TGCTTTCCCTCCTTCCTTCTT 1148 156 miRNA-156 mir-152 160 mir-152 TCAGTGCATGACAGAACTTGG 282 mir-135_1 161 miR-135 (RFAM- TATGGCTTTTTATTCCTATGTGA 1149 Human) mir-135_1 161 mir-135 TATGGCTTTTTATTCCTATGTGAT 283 non-coding RNA in 162 miR-135 (RFAM- TATGGCTTTTTATTCCTATGTGA 1149 rhabdomyosarcoma/ Human) mir-135_2 non-coding RNA in 162 mir-135 TATGGCTTTTTATTCCTATGTGAT 283 rhabdomyosarcoma/ mir-135_2 mir-217 163 mir-217 (human) TACTGCATCAGGAACTGATTGGAT 284 hypothetical miRNA 164 hypothetical TGGCCATAAACTTGTAGTCAT 1150 161 miRNA-161 mir-15a 165 mir-15_Ruvkun TAGCAGCACATAATGGTTTGT 1151 mir-15a 165 mir-15a TAGCAGCACATAATGGTTTGTG 269 let-7g 166 let-7g TGAGGTAGTAGTTTGTACAGT 285 let-7g 166 let-7gL_Ruvkun TGAGGTAGTAGTTTGTACAGTT 1152 hypothetical miRNA 167 hypothetical TGCAAGGATTTTTATGTTTTG 1153 164 miRNA-164 hypothetical miRNA 169 hypothetical TTCCAGTTGCAGCACCTGTAA 1154 166 miRNA-166 hypothetical miRNA 171 hypothetical AGCCAGGTGCCTTCACCTGCT 1155 168_1/similar to miRNA-168 ribosomal protein L5 forkhead box 172 hypothetical TGGCAGCTCTGGCATTTCATA 1156 P2/hypothetical miRNA-169 miRNA-169 hypothetical miRNA 173 hypothetical TGATCTTGCTCTAACACTTGG 1157 170 miRNA-170 glutamate 174 hypothetical TGACAAGTATGTTTTATCGTT 1158 receptor, miRNA-171 ionotropic, AMPA 2/ hypothetical miRNA-171 hypothetical miRNA 175 hypothetical TCCAACTGCAAGAAGTTACT 1159 172 miRNA-172 hypothetical miRNA 176 hypothetical TAGTACGAGAAGAAGGAGGCT 1160 173 miRNA-173 mir-182 177 miR-182* (RFAM- TGGTTCTAGACTTGCCAACTA 1161 Human) mir-182 177 mir-182 TTTGGCAATGGTAGAACTCACA 287 hypothetical miRNA 178 hypothetical TCTCCTTCAACCACCTGAGGT 1162 175 miRNA-175 hypothetical miRNA 179 hypothetical TAGGAGTTTGATATGACATAT 1163 176 miRNA-176 hypothetical 180 hypothetical AGACAAACATGCTACTCTCAC 1164 miRNA-177_1 miRNA-177 hypothetical miRNA 181 hypothetical TAGCCTATCTCCGAACCTTCA 1165 178 miRNA-178 hypothetical miRNA 182 hypothetical TGAAAGGCACTTTGTCCAATT 1166 179 miRNA-179 hypothetical miRNA 184 hypothetical TCACCTGCTCTGGAAGTAGTT 1167 181 miRNA-181 mir-148a 185 mir-148a TCAGTGCACTACAGAACTTTGT 288 hypothetical miRNA 188 hypothetical TGATGGCCAGCTGAGCAGCTC 1168 185 miRNA-185 hypothetical 189 hypothetical AGACAAACATGCTACTCTCAC 1164 miRNA-177_2/ miRNA-177 hypothetical miRNA 186 mir-181c 190 mir-181c AACATTCAACCTGTCGGTGAGT 290 hypothetical miRNA 191 hypothetical TGGTGAGGGGAATGAAAAGTA 1169 188 miRNA-188 mir-100_1 945 mir-100 AACCCGTAGATCCGAACTTGTG 275 mir-101_1 946 mir-101 TACAGTACTGTGATAACTGA 265 mir-101_1 946 miR-101 (RFAM- TACAGTACTGTGATAACTGAAG 1170 Human) mir-101_3 947 mir-101 TACAGTACTGTGATAACTGA 265 mir-101_3 947 miR-101 (RFAM- TACAGTACTGTGATAACTGAAG 1170 Human) mir-29b_2 948 miR-29b (RFAM- TAGCACCATTTGAAATCAGT 1172 Human) mir-29b_2 948 miR-29b (RFAM-M. mu.) TAGCACCATTTGAAATCAGTGT 1173 mir-29b_2 948 mir-29b TAGCACCATTTGAAATCAGTGTT 195 mir-29b_1 949 miR-29b (RFAM- TAGCACCATTTGAAATCAGT 1172 Human) mir-29b_1 949 miR-29b (RFAM-M. mu.) TAGCACCATTTGAAATCAGTGT 1173 mir-29b_1 949 mir-29b TAGCACCATTTGAAATCAGTGTT 195 mir-103_1 950 mir-103 AGCAGCATTGTACAGGGCTATGA 225 mir-106 951 mir-106 (human) AAAAGTGCTTACAGTGCAGGTAGC 230 mir-107 952 mir-107 AGCAGCATTGTACAGGGCTATCA 229 mir-16_1 953 mir-16 TAGCAGCACGTAAATATTGGCG 196 mir-16_1 953 mir-16_Ruvkun TAGCAGCACGTAAATATTGGCGT 1176 mir-16_3 954 mir-16 TAGCAGCACGTAAATATTGGCG 196 mir-16_3 954 mir-16_Ruvkun TAGCAGCACGTAAATATTGGCGT 1176 mir-18 955 mir-18 TAAGGTGCATCTAGTGCAGATA 262 mir-18 955 mir-18_Ruvkun TAAGGTGCATCTAGTGCAGATAG 1177 mir-19a 956 mir-19a TGTGCAAATCTATGCAAAACTGA 268 mir-19b_1 957 mir-19b* (Michael AGTTTTGCAGGTTTGCATCCAGC 1179 et al) mir-19b_1 957 mir-19b TGTGCAAATCCATGCAAAACTGA 241 mir-19b_2 958 mir-19b TGTGCAAATCCATGCAAAACTGA 241 mir-21 959 mir-21 TAGCTTATCAGACTGATGTTGA 236 mir-23a 960 mir-23a ATCACATTGCCAGGGATTTCC 289 mir-24_2 961 mir-24 TGGCTCAGTTCAGCAGGAACAG 264 mir-17/mir-91 962 mir-17 (human, ACTGCAGTGAAGGCACTTGT 1180 rat) mir-17/mir-91 962 mir-91_Ruvkun CAAAGTGCTTACAGTGCAGGTAG 1181 mir-17/mir-91 962 mir-17as/mir-91 CAAAGTGCTTACAGTGCAGGTAGT 204 mir-92_1 963 miR-92 (RFAM-M. mu.) TATTGCACTTGTCCCGGCCTG 1182 mir-92_1 963 mir-92 TATTGCACTTGTCCCGGCCTGT 216 mir-96 964 mir-96 TTTGGCACTAGCACATTTTTGC 203 mir-96 964 miR-96 (RFAM-M. mu.) TTTGGCACTAGCACATTTTTGCT 1183 mir-30a 965 mir-30a CTTTCAGTCGGATGTTTGCAGC 193 mir-30a 965 miR-30a-s TGTAAACATCCTCGACTGGAAGC 1184 mir-98 966 mir-98 TGAGGTAGTAAGTTGTATTGTT 257 mir-104 967 miR-104 TCAACATCAGTCTGATAAGCTA 335 (Mourelatos) (Mourelatos) mir-105 968 miR-105 TCAAATGCTCAGACTCCTGT 1185 (Mourelatos) (Mourelatos) mir-27 969 miR-27 TTCACAGTGGCTAAGTTCC 1186 (Mourelatos) (Mourelatos) mir-27 969 miR-27a (RFAM-M. mu.) TTCACAGTGGCTAAGTTCCGC 1187 (Mourelatos) mir-27 969 miR-27a (RFAM- TTCACAGTGGCTAAGTTCCGCC 1188 (Mourelatos) Human) mir-92_2 970 miR-92 (RFAM-M. mu.) TATTGCACTTGTCCCGGCCTG 1182 mir-92_2 970 mir-92 TATTGCACTTGTCCCGGCCTGT 216 mir-93 971 miR-93 AAAGTGCTGTTCGTGCAGGTAG 1189 (Mourelatos) (Mourelatos) mir-93 971 miR-93 (Tuschl) CAAAGTGCTGTTCGTGC 1190 (Mourelatos) mir-93 971 miR-93 (RFAM-M. mu.) CAAAGTGCTGTTCGTGCAGGTAG 1191 (Mourelatos) mir-95 972 miR-95 TTCAACGGGTATTTATTGAGCA 1192 (Mourelatos) (Mourelatos) mir-99 973 miR-99 AACCCGTAGATCCGATCTTGTG 1193 (Mourelatos) (Mourelatos) mir-99 973 miR-99a (Tuschl) ACCCGTAGATCCGATCTTGT 1194 (Mourelatos) mir-25 974 miR-25 (Tuschl) CATTGCACTTGTCTCGGTCTGA 1195 mir-28 975 miR-28 (Tuschl) AAGGAGCTCACAGTCTATTGAG 1196 mir-31 976 miR-31 (RFAM-M. mu.) AGGCAAGATGCTGGCATAGCTG 1197 mir-31 976 miR-31 (Tuschl) GGCAAGATGCTGGCATAGCTG 1198 mir-32 977 miR-32 (Tuschl) TATTGCACATTACTAAGTTGC 1199 mir-149 978 miR-149 TCTGGCTCCGTGTCTTCACTCC 1200 mir-30c_2 979 mir-30c TGTAAACATCCTACACTCTCAGC 280 mir-30c_2 979 mir-30c_Ruvkun TGTAAACATCCTACACTCTCAGCT 1129 mir-99b 980 miR-99b CACCCGTAGAACCGACCTTGCG 1201 MiR-125a 981 miR-125a TCCCTGAGACCCTTTAACCTGTG 1202 MiR-125b_2 982 mir-125b TCCCTGAGACCCTAACTTGTGA 258 mir-26a_2 983 miR-26a (Michael TTCAAGTAATCCAGGATAGGC 1203 et al) mir-26a_2 983 mir-26a TTCAAGTAATCCAGGATAGGCT 226 mir-127 984 mir-127_Ruvkun TCGGATCCGTCTGAGCTTGG 1204 mir-127 984 miR-127 TCGGATCCGTCTGAGCTTGGCT 1205 mir-136 985 miR-136 ACTCCATTTGTTTTGATGATGGA 1206 mir-154 986 miR-154 TAGGTTATCCGTGTTGCCTTCG 1207 mir-26a_1 987 miR-26a (Michael TTCAAGTAATCCAGGATAGGC 1203 et al) mir-26a_1 987 mir-26a TTCAAGTAATCCAGGATAGGCT 226 mir_186 988 miR-186 CAAAGAATTCTCCTTTTGGGCTT 1208 mir_198 989 mir-198 GGTCCAGAGGGGAGATAGG 1209 mir_191 990 mir-191 CAACGGAATCCCAAAAGCAGCT 1210 mir_191 990 mir-191_Ruvkun CAACGGAATCCCAAAAGCAGCTGT 1211 mir_206 991 mir-206 TGGAATGTAAGGAAGTGTGTGG 1212 mir-94/mir-106b 992 miR-94 AAAGTGCTGACAGTGCAGAT 1213 mir-94/mir-106b 992 miR-106b (RFAM-M. mu.) TAAAGTGCTGACAGTGCAGAT 1214 mir_184 993 miR-184 TGGACGGAGAACTGATAAGGGT 1215 mir_195 994 miR-195 TAGCAGCACAGAAATATTGGC 1216 mir_193 995 miR-193 AACTGGCCTACAAAGTCCCAG 1217 mir_185 996 miR-185 TGGAGAGAAAGGCAGTTC 1218 mir_188 997 miR-188 CATCCCTTGCATGGTGGAGGGT 1219 mir_197 998 miR-197a TTCACCACCTTCTCCACCCAGC 1220 mir_194_1 999 miR-194 TGTAACAGCAACTCCATGTGGA 1221 mir_208 1000 miR-208 ATAAGACGAGCAAAAAGCTTGT 1222 mir_194_2 1001 miR-194 TGTAACAGCAACTCCATGTGGA 1221 mir_139 1002 miR-139 TCTACAGTGCACGTGTCT 1223 mir-200b 1003 miR-200a (RFAM- CTCTAATACTGCCTGGTAATGATG 1224 Human) mir-200b 1003 miR-200b (Michael TAATACTGCCTGGTAATGATGA 1225 et al) mir-200b 1003 miR-200b TAATACTGCCTGGTAATGATGAC 1226 mir-200a 1004 miR-200a TAACACTGTCTGGTAACGATG 1227 mir-200a 1004 miR-200a (RFAM-M. mu.) TAACACTGTCTGGTAACGATGT 1228 mir-240* (Kosik) 1005 mir-240* (Kosik) TCAAGAGCAATAACGAAAAATGT 1229 mir-232* (Kosik) 1006 mir-232* (Kosik) CTGGCCCTCTCTGCCCTTCCGT 1230 mir-227* 1007 mir-226* (Kosik) ACTGCCCCAGGTGCTGCTGG 1231 (Kosik)/mir-226* (Kosik) mir-227* 1007 mir-324-3p_Ruvkun CCACTGCCCCAGGTGCTGCTGG 1232 (Kosik)/mir-226* (Kosik) mir-227* 1007 mir-227* (Kosik) CGCATCCCCTAGGGCATTGGTGT 1233 (Kosik)/mir-226* (Kosik) mir-244* (Kosik) 1008 mir-244* (Kosik) TCCAGCATCAGTGATTTTGTTGA 1234 mir-224* (Kosik) 1009 mir-224* (Kosik) GCACATTACACGGTCGACCTCT 1235 mir-248* (Kosik) 1010 mir-248* (Kosik) TCTCACACAGAAATCGCACCCGTC 1236 ribosomal protein 1011 hypothetical AGCCAGGTGCCTTCACCTGCT 1155 L5/hypothetical miRNA-168 miRNA 168_2 hypothetical 1012 hypothetical AGACAAACATGCTACTCTCAC 1164 miRNA-177_3 miRNA-177 mir-138_3 1013 mir-138 AGCTGGTGTTGTGAATC 256 mir-138_3 1013 mir-138_Ruvkun AGCTGGTGTTGTGAATCAGGCCG 1127 mir-138_4 1014 mir-138 AGCTGGTGTTGTGAATC 256 mir-181b_2 1015 mir-181b AACATTCATTGCTGTCGGTGGGTT 260 mir-219_1 1016 mir-219 TGATTGTCCAAACGCAATTCT 271 mir-105_2 1017 miR-105 TCAAATGCTCAGACTCCTGT 1185 (Mourelatos) hypothetical miRNA 1018 hypothetical TTACAGCAATCCAGTAATGAT 1138 120_2 miRNA-120 cezanne 2/ 1019 hypothetical TCCTGTCAGACTTTGTTCGGT 1237 hypothetical miRNA-180 miRNA-180 mir-103_2 1020 mir-103 AGCAGCATTGTACAGGGCTATGA 225 mir-147 (Sanger) 1021 miR-147 (RFAM- GTGTGTGGAAATGCTTCTGC 1238 Human) mir-224 (Sanger) 1022 miR-224 (RFAM- CAAGTCACTAGTGGTTCCGTTTA 1239 Human) mir-134 (Sanger) 1023 miR-134 (RFAM- TGTGACTGGTTGACCAGAGGG 1240 Human) mir-146 (Sanger) 1024 miR-146 (RFAM- TGAGAACTGAATTCCATGGGTT 1241 Human) mir-150 (Sanger) 1025 miR-150 (RFAM- TCTCCCAACCCTTGTACCAGTG 1242 Human) mir-30e (RFAM/mmu) 1026 miR-30e (RFAM-M. mu.) TGTAAACATCCTTGACTGGA 1243 mir-30e (RFAM/mmu) 1026 miR-97 (Michael TGTAAACATCCTTGACTGGAAG 1244 et al) mir-296 (RFAM/mmu) 1027 miR-296 (RFAM-M. mu.) AGGGCCCCCCCTCAATCCTGT 1245 mir-299 (RFAM/mmu) 1028 miR-299 (RFAM-M. mu.) TGGTTTACCGTCCCACATACAT 1246 mir-301 (RFAM/mmu) 1029 miR-301 (RFAM-M. mu.) CAGTGCAATAGTATTGTCAAAGC 1247 mir-301 (RFAM/mmu) 1029 mir-301_Ruvkun CAGTGCAATAGTATTGTCAAAGCAT 1248 mir-302 (RFAM/mmu) 1030 miR-302 (RFAM-M. mu.) TAAGTGCTTCCATGTTTTGGTGA 1249 mir-34a (RFAM/mmu) 1031 mir-34c (RFAM) AGGCAGTGTAGTTAGCTGATTG 1250 mir-34a (RFAM/mmu) 1031 miR-34a (RFAM-M. mu.) AGGCAGTGTAGTTAGCTGATTGC 1251 mir_320 1032 miR-320 AAAAGCTGGGTTGAGAGGGCGAA 1252 mir-321_1 1033 miR-321-1 TAAGCCAGGGATTGTGGGTTC 1253 mir-135b (Ruvkun) 1034 mir-135b (Ruvkun) TATGGCTTTTCATTCCTATGTG 1254 mir-151* (Ruvkun) 1035 mir-151 (human) ACTAGACTGAAGCTCCTTGAGG 1255 mir-151* (Ruvkun) 1035 mir-151* (Ruvkun) TCGAGGAGCTCACAGTCTAGTA 1256 mir-340 (Ruvkun) 1036 mir-340 (Ruvkun) TCCGTCTCAGTTACTTTATAGCC 1257 mir-331 (Ruvkun) 1037 mir-331 (Ruvkun) GCCCCTGGGCCTATCCTAGAA 1258 mir_200c (RFAM) 1038 mir-200c (RFAM) AATACTGCCGGGTAATGATGGA 1259 mir_34b (RFAM) 1039 mir-34b (RFAM) AGGCAGTGTCATTAGCTGATTG 1260 mir_339_1 (RFAM) 1040 mir-339 (RFAM) TCCCTGTCCTCCAGGAGCTCA 1261 mir_339_2 (RFAM) 1041 mir-339 (RFAM) TCCCTGTCCTCCAGGAGCTCA 1261 mir-325 (Ruvkun) 1042 mir-325 (human) CCTAGTAGGTGTCCAGTAAGTGT 1262 mir-326 (Ruvkun) 1043 miR-326 (Ruvkun) CCTCTGGGCCCTTCCTCCAG 1263 mir-326 (Ruvkun) 1044 mir-326 (human) CCTCTGGGCCCTTCCTCCAGC 1264 mir-329-1 (Ruvkun) 1045 mir-329 (human) AACACACCTGGTTAACCTCTTT 1265 mir-329-2 (Ruvkun) 1046 mir-329 (human) AACACACCTGGTTAACCTCTTT 1265 mir-330 (Ruvkun) 1047 mir-330 (human) GCAAAGCACACGGCCTGCAGAGA 1266 mir-337 (Ruvkun) 1048 mir-337 (human) TCCAGCTCCTATATGATGCCTTT 1267 mir-345 (Ruvkun) 1049 mir-345 (human) TGCTGACTCCTAGTCCAGGGC 1268 mir-346 (Ruvkun) 1050 mir-346 (human) TGTCTGCCCGCATGCCTGCCTCT 1269 mir-187 1051 miR-187 (RFAM- TCGTGTCTTGTGTTGCAGCCG 1270 Human) mir-187 1051 mir-187 TCGTGTCTTGTGTTGCAGCCGG 276 miR-24-1 1052 miR-189 (RFAM- GTGCCTACTGAGCTGATATCAGT 1271 Human) miR-24-1 1052 mir-24 TGGCTCAGTTCAGCAGGAACAG 264 mir-215 1053 mir-215 ATGACCTATGAATTGACAGAC 278

A list of mouse pri-miRNAs and the mature miRNAs predicted to derive from them is shown in Table 61. “Pri-miRNA name” indicates the gene name for each of the pri-miRNAs. Also given in table 61 are the name and sequence of the mature miRNA derived from the pri-miRNA. Mature miRNA sequences from pri-miRNA precursors have been proposed by several groups; consequently, for a given pri-miRNA sequence, several miRNAs may be disclosed and given unique names, and thus a given pri-miRNA sequence may occur repeatedly in the table. The sequences are written in the 5′ to 3′ direction and are represented in the DNA form. It is understood that a person having ordinary skill in the art would be able to convert the sequence of the targets to their RNA form by simply replacing the thymidine (T) with uracil (U) in the sequence.

TABLE 61 Mouse pri-miRNA sequences and the corresponding mature miRNAs SEQ SEQ ID ID Pri-miRNA name NO Mature miRNA name Mature miRNA sequence NO mir-26b 1273 miR-99 TTCAAGTAATTCAGGATAGGT 1147 (Mourelatos) mir-26b 1273 miR-199-as TTCAAGTAATTCAGGATAGGTT 281 mir-30a 1274 miR-199-as CTTTCAGTCGGATGTTTGCAGC 193 mir-30a 1274 miR-26b (RFAM- TGTAAACATCCTCGACTGGAAGC 1184 Human) mir-30c_1 1275 miR-32 (Tuschl) TGTAAACATCCTACACTCTCAGC 280 mir-30c_1 1275 let-7c_Ruvkun TGTAAACATCCTACACTCTCAGCT 1129 mir-128a 1276 mir-214 TCACAGTGAACCGGTCTCTTT 1073 mir-128a 1276 miR-29b (RFAM- TCACAGTGAACCGGTCTCTTTT 200 Human) mir-29b_1 1277 mir-196 TAGCACCATTTGAAATCAGT 1172 mir-29b_1 1277 hypothetical TAGCACCATTTGAAATCAGTGT 1173 miRNA-079 mir-29b_1 1277 mir-30c TAGCACCATTTGAAATCAGTGTT 195 mir-29c 1278 mir-131_Ruvkun CTAGCACCATTTGAAATCGGTT 232 mir-29c 1278 hypothetical TAGCACCATTTGAAATCGGTTA 1100 miRNA-033 mir-123/mir-126 1279 mir-326 (rodent) CATTATTACTTTTGGTACGCG 205 mir-123/mir-126 1279 mir-126 TCGTACCGTGAGTAATAATGC 1076 mir-130a 1280 mir-23a CAGTGCAATGTTAAAAGGGC 233 mir-130a 1280 hypothetical CAGTGCAATGTTAAAAGGGCAT 1101 miRNA-040 mir-1d_1 1281 mir-132 TGGAATGTAAAGAAGTATGTA 1083 mir-1d_1 1281 mir-124a (Kosik) TGGAATGTAAAGAAGTATGTAT 213 mir-1d_1 1281 miR-200b TGGAATGTAAAGAAGTATGTATT 1134 mir-124a_3 1282 mir-100 TAAGGCACGCGGTGAATGCCA 1104 mir-124a_3 1282 mir-212 TTAAGGCACGCGGTGAATGCCA 235 mir-124a_3 1282 let-7a TTAAGGCACGCGGTGAATGCCAA 1105 mir-133a_2 1283 miR-189 (RFAM- TTGGTCCCCTTCAACCAGCTGT 255 Human) mir-124a_2 1284 mir-181c TAAGGCACGCGGTGAATGCCA 1104 mir-124a_2 1284 mir-108 TTAAGGCACGCGGTGAATGCCA 235 mir-124a_2 1284 mir-239* (Kosik) TTAAGGCACGCGGTGAATGCCAA 1105 mir-131_1/mir-9 1285 mir-325 (rodent) TAAAGCTAGATAACCGAAAGT 211 mir-131_1/mir-9 1285 mir-19b TAAAGCTAGATAACCGAAAGTA 1080 mir-131_1/mir-9 1285 mir-124a_Ruvkun TCTTTGGTTATCTAGCTGTATGA 1081 mir-15b 1286 mir-152 TAGCAGCACATCATGGTTTAC 1115 mir-15b 1286 hypothetical TAGCAGCACATCATGGTTTACA 246 miRNA-111 mir-16_3 1287 miR-104 TAGCAGCACGTAAATATTGGCG 196 (Mourelatos) mir-16_3 1287 mir-128a TAGCAGCACGTAAATATTGGCGT 1176 mir-137 1288 mir-30b TATTGCTTAAGAATACGCGTAG 270 mir-101_1 1289 mir-18 TACAGTACTGTGATAACTGA 265 mir-101_1 1289 mir-128b TACAGTACTGTGATAACTGAAG 1170 mir-29a 1291 miR-27a (RFAM-M. mu.) CTAGCACCATCTGAAATCGGTT 247 mir-29a 1291 mir-153 TAGCACCATCTGAAATCGGTTA 1116 mir-29b_2 1292 mir-138_Ruvkun TAGCACCATTTGAAATCAGT 1172 mir-29b_2 1292 hypothetical TAGCACCATTTGAAATCAGTGT 1173 miRNA-075 mir-29b_2 1292 miR-30a-s TAGCACCATTTGAAATCAGTGTT 195 mir-148a 1293 miR-1d (Tuschl) TCAGTGCACTACAGAACTTTGT 288 mir-141 1294 mir-16_Ruvkun AACACTGTCTGGTAAAGATGG 261 mir-131_3/mir-9 1295 mir-124a (Kosik) TAAAGCTAGATAACCGAAAGT 211 mir-131_3/mir-9 1295 mir-7b (rodent) TAAAGCTAGATAACCGAAAGTA 1080 mir-131_3/mir-9 1295 mir-19a TCTTTGGTTATCTAGCTGTATGA 1081 mir-23a 1296 miR-1 (RFAM) ATCACATTGCCAGGGATTTCC 289 mir-24_2 1297 mir-124a_Ruvkun TGGCTCAGTTCAGCAGGAACAG 264 mir-140 1298 miR-199b (mouse) AGTGGTTTTACCCTATGGTAG 192 mir-140 1298 mir-205 TACCACAGGGTAGAACCACGGA 1065 mir-140 1298 mir-26b TACCACAGGGTAGAACCACGGACA 1066 let-7a_4 1299 mir-16_Ruvkun TGAGGTAGTAGGTTGTATAGTT 222 mir-125b_1 1300 mir-131_Ruvkun TCCCTGAGACCCTAACTTGTGA 258 mir-26a_1 1301 mir-29b TTCAAGTAATCCAGGATAGGC 1203 mir-26a_1 1301 hypothetical TTCAAGTAATCCAGGATAGGCT 226 miRNA-154 let-7i 1302 hypothetical TGAGGTAGTAGTTTGTGCT 209 miRNA-179 let-7i 1302 miR-1d (Tuschl) TGAGGTAGTAGTTTGTGCTGTT 1078 mir-21 1303 mir-125b TAGCTTATCAGACTGATGTTGA 236 mir-22 1304 mir-131 AAGCTGCCAGTTGAAGAACTGT 215 mir-142 1305 mir-131_Ruvkun CATAAAGTAGAAAGCACTAC 217 mir-142 1305 hypothetical TGTAGTGTTTCCTACTTTATGG 1086 miRNA-105 mir-142 1305 mir-218 TGTAGTGTTTCCTACTTTATGGA 1087 mir-144 1306 mir-26a TACAGTATAGATGATGTACTAG 237 mir-152 1307 miR-99a (Tuschl) TCAGTGCATGACAGAACTTGG 282 mir-153_2 1308 mir-29c TTGCATAGTCACAAAAGTGA 201 let-7a_1 1309 mir-16 TGAGGTAGTAGGTTGTATAGTT 222 let-7d 1310 mir-144 AGAGGTAGTAGGTTGCATAGT 245 let-7d 1310 hypothetical AGAGGTAGTAGGTTGCATAGTT 1113 miRNA-171 let-7d 1310 miR-204 (Tuschl) CTATACGACCTGCTGCCTTTCT 1114 let-7f_1 1311 miR-9 TGAGGTAGTAGATTGTATAGT 1098 let-7f_1 1311 hypothetical TGAGGTAGTAGATTGTATAGTT 231 miRNA-138 mir-23b 1312 mir-1d ATCACATTGCCAGGGATTACCAC 208 miR-24-1 1313 mir-124a (Kosik) GTGCCTACTGAGCTGATATCAGT 1271 miR-24-1 1313 hypothetical TGGCTCAGTTCAGCAGGAACAG 264 miRNA-070 mir-27b 1314 miR-29c (Tuschl) TTCACAGTGGCTAAGTTCTG 202 mir-27b 1314 mir-135 TTCACAGTGGCTAAGTTCTGC 1059 mir-131_2/mir-9 1315 mir-107 TAAAGCTAGATAACCGAAAGT 211 mir-131_2/mir-9 1315 miR-224 (RFAM- TAAAGCTAGATAACCGAAAGTA 1080 mouse) mir-131_2/mir-9 1315 mir-124a TCTTTGGTTATCTAGCTGTATGA 1081 mir-15a 1316 miR-20 (RFAM- TAGCAGCACATAATGGTTTGT 1151 Human) mir-15a 1316 miR-92 (RFAM- TAGCAGCACATAATGGTTTGTG 269 M. mu.) mir-16_1 1317 mir-98 TAGCAGCACGTAAATATTGGCG 196 mir-16_1 1317 mir-30c_Ruvkun TAGCAGCACGTAAATATTGGCGT 1176 mir-124a_1 1318 miR-132 (RFAM- TAAGGCACGCGGTGAATGCCA 1104 Human) mir-124a_1 1318 miR-140-as TTAAGGCACGCGGTGAATGCCA 235 mir-124a_1 1318 hypothetical TTAAGGCACGCGGTGAATGCCAA 1105 miRNA-181 mir-18 1319 mir-124a TAAGGTGCATCTAGTGCAGATA 262 mir-18 1319 miR-27 TAAGGTGCATCTAGTGCAGATAG 1177 (Mourelatos) mir-20 1320 mir-23b TAAAGTGCTTATAGTGCAGGTA 1126 mir-20 1320 mir-199a TAAAGTGCTTATAGTGCAGGTAG 254 mir-30b 1321 miR-31 (Tuschl) TGTAAACATCCTACACTCAGC 266 mir-30b 1321 mir-18_Ruvkun TGTAAACATCCTACACTCAGCT 1137 mir-30d 1322 miR-186 TGTAAACATCCCCGACTGGAAG 240 mir-30d 1322 let-7i_Ruvkun TGTAAACATCCCCGACTGGAAGCT 1108 let-7b 1323 mir-135 TGAGGTAGTAGGTTGTGTGGTT 212 let-7b 1323 mir-133a TGAGGTAGTAGGTTGTGTGGTTT 1082 let7c_2 1324 let-7d* (RFAM-M. mu.) TGAGGTAGTAGGTTGTATGGTT 250 let7c_2 1324 hypothetical TGAGGTAGTAGGTTGTATGGTTT 1120 miRNA-170 let-7c_1 1325 let-7d TGAGGTAGTAGGTTGTATGGTT 250 let-7c_1 1325 miR-135 (RFAM- TGAGGTAGTAGGTTGTATGGTTT 1120 Human) mir-99 1326 miR-203 (Tuschl) AACCCGTAGATCCGATCTTGTG 1193 (Mourelatos) mir-99 1326 mir-34 ACCCGTAGATCCGATCTTGT 1194 (Mourelatos) LOC 114614 1327 mir-187 TTAATGCTAATTGTGATAGGGG 1459 containing miR- 155/hypothetical miRNA-071 let-7e 1328 let-7a TGAGGTAGGAGGTTGTATAGT 249 mir-1d_2 1329 miR-10b (Michael TGGAATGTAAAGAAGTATGTA 1083 et al) mir-1d_2 1329 miR-139 TGGAATGTAAAGAAGTATGTAT 213 mir-1d_2 1329 mir-124a TGGAATGTAAAGAAGTATGTATT 1134 mir-133a_1 1330 mir-24 TTGGTCCCCTTCAACCAGCTGT 255 mir-143 1331 miR-15b (Michael TGAGATGAAGCACTGTAGCTC 1088 et al) mir-143 1331 mir-253* (Kosik) TGAGATGAAGCACTGTAGCTCA 220 mir-145 1332 mir-148b GTCCAGTTTTCCCAGGAATCC 1122 mir-145 1332 let-7f GTCCAGTTTTCCCAGGAATCCCTT 252 mir-122a 1333 miR-172 (RFAM-M. mu.) TGGAGTGTGACAATGGTGTTTG 1084 mir-122a 1333 mir-124a_Ruvkun TGGAGTGTGACAATGGTGTTTGT 214 mir-19b_2 1334 mir-22 TGTGCAAATCCATGCAAAACTGA 241 let-7f_2 1335 hypothetical TGAGGTAGTAGATTGTATAGT 1098 miRNA-137 let-7f_2 1335 mir-131 TGAGGTAGTAGATTGTATAGTT 231 mir-26a_2 1336 mir-29a_Ruvkun TTCAAGTAATCCAGGATAGGC 1203 mir-26a_2 1336 hypothetical TTCAAGTAATCCAGGATAGGCT 226 miRNA-153 mir-127 1337 mir-103 TCGGATCCGTCTGAGCTTGG 1204 mir-127 1337 mir-17as/mir-91 TCGGATCCGTCTGAGCTTGGCT 1205 mir-136 1338 mir-91_Ruvkun ACTCCATTTGTTTTGATGATGGA 1206 mir-154 1339 mir-17-3p (mouse) TAGGTTATCCGTGTTGCCTTCG 1207 mir-149 1340 let-7gL_Ruvkun TCTGGCTCCGTGTCTTCACTCC 1200 mir-30c_2 1341 miR-31 (RFAM- TGTAAACATCCTACACTCTCAGC 280 M. mu.) mir-30c_2 1341 let-7c TGTAAACATCCTACACTCTCAGCT 1129 mir-99b 1342 mir-101b (rodent) CACCCGTAGAACCGACCTTGCG 1201 MiR-125a 1343 mir-106 (mouse) TCCCTGAGACCCTTTAACCTGTG 1202 MiR-125b_2 1344 miR-9 TCCCTGAGACCCTAACTTGTGA 258 mir-221 1345 miR-200a (RFAM- AGCTACATTGTCTGCTGGGTTT 1106 Human) mir-221 1345 miR-26a (Michael AGCTACATTGTCTGCTGGGTTTC 238 et al) mir-203 1346 mir-10b GTGAAATGTTTAGGACCACTAG 197 mir-203 1346 mir-128 (Kosik) TGAAATGTTTAGGACCACTAG 1068 mir-203 1346 mir-204 TGAAATGTTTAGGACCACTAGA 1069 let-7g 1347 hypothetical TGAGGTAGTAGTTTGTACAGT 285 miRNA-176 let-7g 1347 mir-1d TGAGGTAGTAGTTTGTACAGTT 1152 mir-101_3 1348 miR-200a TACAGTACTGTGATAGCTGAAG 1460 mir-106 1349 miR-200a (RFAM- CAAAGTGCTAACAGTGCAGGTA 1461 M. mu.) mir-17/mir-91 1350 mir-123/mir-126as ACTGCAGTGAGGGCACTTGT 1462 mir-17/mir-91 1350 mir-227* (Kosik) CAAAGTGCTTACAGTGCAGGTAG 1181 mir-17/mir-91 1350 miR-195 CAAAGTGCTTACAGTGCAGGTAGT 204 mir-199b 1351 mir-226* (Kosik) CCCAGTGTTTAGACTACCTGTTC 1463 mir-199b 1351 mir-217 (rodent) TACAGTAGTCTGCACATTGGTT 1118 hypothetical 1352 mir-324-3p_Ruvkun AGAGGTATAGCGCATGGGAAGA 1464 miRNA 105 hypothetical 1352 miR-127 TTCCTATGCATATACTTCTTT 1132 miRNA 105 mir-211 1353 mir-244* (Kosik) TTCCCTTTGTCATCCTTTGCCT 1465 mir-217 1354 mir-224* (Kosik) TACTGCATCAGGAACTGACTGGAT 1466 mir-224 (Sanger) 1355 mir-248* (Kosik) TAAGTCACTAGTGGTTCCGTTTA 1467 mir-7_3 1356 mir-138 TGGAAGACTTGTGATTTTGTT 1468 mir-325 (Ruvkun) 1357 mir-138_Ruvkun CCTAGTAGGTGCTCAGTAAGTGT 1469 mir-326 (Ruvkun) 1358 mir-181b CCTCTGGGCCCTTCCTCCAG 1263 mir-326 (Ruvkun) 1358 miR-298 CCTCTGGGCCCTTCCTCCAGT 1470 mir-329-1 1359 mir-103 AACACACCCAGCTAACCTTTTT 1471 (Ruvkun) mir-330 (Ruvkun) 1360 miR-134 (RFAM- GCAAAGCACAGGGCCTGCAGAGA 1472 Human) mir-337 (Ruvkun) 1361 miR-146 (RFAM- TTCAGCTCCTATATGATGCCTTT 1473 Human) mir-345 (Ruvkun) 1362 miR-30e (RFAM- TGCTGACCCCTAGTCCAGTGC 1474 M. mu.) mir-346 (Ruvkun) 1363 miR-97 (Michael TGTCTGCCCGAGTGCCTGCCTCT 1475 et al) mir-151* (Ruvkun) 1364 miR-193 ACTAGACTGAGGCTCCTTGAGG 1476 mir-151* (Ruvkun) 1364 mir-340 (Ruvkun) CTAGACTGAGGCTCCTTGAGG 1477 mir-151* (Ruvkun) 1364 miR-299 (RFAM- TCGAGGAGCTCACAGTCTAGTA 1256 M. mu.) mir_34b (RFAM) 1365 mir-331 (Ruvkun) TAGGCAGTGTAATTAGCTGATTG 1478 glutamate 1366 miR-143 (Michael TGTTATAGTATTCCACCTACC 1060 receptor, et al) ionotrophic, AMPA 3/hypothetical miRNA-033 mir-34 1367 mir-138 TGGCAGTGTCTTAGCTGGTTGT 194 mir-34 1367 mir-30a TGGCAGTGTCTTAGCTGGTTGTT 1067 mir-7_1/mir-7_1* 1368 mir-191_Ruvkun CAACAAATCACAGTCTGCCATA 1070 mir-7_1/mir-7_1* 1368 mir-29b TGGAAGACTAGTGATTTTGTT 198 mir-10b 1369 mir-210 CCCTGTAGAACCGAATTTGTGT 1071 mir-10b 1369 miR-29b (RFAM- TACCCTGTAGAACCGAATTTGT 199 M. mu.) mir-10b 1369 mir-34b (mouse) TACCCTGTAGAACCGAATTTGTG 1072 mir-132 1370 mir-130a TAACAGTCTACAGCCATGGTCG 1077 mir-132 1370 miR-196 (Tuschl) TAACAGTCTACAGCCATGGTCGC 206 mir-108_1 1371 mir-130 (Kosik) ATAAGGATTTTTAGGGGCATT 207 mir-212 1372 miR-1 (RFAM) TAACAGTCTCCAGTCACGGCC 210 hypothetical 26 mir-143 TGGGCAAGAGGACTTTTTAAT 1079 miRNA 023 mir-214 37 mir-15b ACAGCAGGCACAGACAGGCAG 219 hypothetical 43 mir-145 TGTCAACAAAACTGCTTACAA 1092 miRNA 040 hypothetical 1373 miR-145 (Michael TGACAGGAAATCTTTGAGAGG 1094 miRNA 043 et al) mir-205 1374 mir-101 TCCTTCATTCCACCGGAGTCTG 224 mir-33a 1375 miR-29b (RFAM- GTGCATTGTAGTTGCATTG 227 M. mu.) mir-196_2 1376 mir-7-1*_Ruvkun TAGGTAGTTTCATGTTGTTGG 1097 mir-196_2 1376 mir-148a TAGGTAGTTTCATGTTGTTGGG 228 hypothetical 1377 mir-122a TTGCATGCCCTATTGATTCTC 1099 miRNA 055 hypothetical 1378 miR-122a,b TGTCAGATGCTTAATGTTCTT 1102 miRNA 058 (Tuschl) mir-218_1 1379 mir-140 TTGTGCTTGATCTAACCATGT 234 mir-218_1 1379 mir-196 TTGTGCTTGATCTAACCATGTG 1103 mir-222 1380 miR-200b (Michael AGCTACATCTGGCTACTGGGTCT 1107 et al) mir-222 1380 let-7i AGCTACATCTGGCTACTGGGTCTC 239 mir-128b 1381 mir-142 TCACAGTGAACCGGTCTCTTT 1073 mir-128b 1381 hypothetical TCACAGTGAACCGGTCTCTTTC 242 miRNA-023 mir-219_2 1382 mir-30b_Ruvkun TGATTGTCCAAACGCAATTCT 271 hypothetical 1383 mir-19b TCACATTTGCCTGCAGAGATT 1109 miRNA 070 mir-129_2 1384 miR-196 (Tuschl) AAGCCCTTACCCCAAAAAGCAT 1110 mir-129_2 1384 mir-128 (Kosik) CTTTTTGCGGTCTGGGCTTGC 243 mir-129_2 1384 miR-142-as CTTTTTGCGGTCTGGGCTTGCT 1111 mir-133b 1385 miR-142as TTGGTCCCCTTCAACCAGCTA 244 (Michael et al) hypothetical 78 let-7f TGGTTAAAATATTAATGGGGC 1112 miRNA 075 hypothetical 1386 let-7f (Michael TGATATGTTTGATATTGGG 1117 miRNA 079 et al) mir-204 1387 let-7d_Ruvkun TTCCCTTTGTCATCCTATGCCT 251 mir-204 1387 miR-10b (Tuschl) TTCCCTTTGTCATCCTATGCCTG 1121 mir-213/mir- 1388 mir-137 AACATTCAACGCTGTCGGTGAG 1096 181a_2 mir-213/mir- 1388 hypothetical AACATTCAACGCTGTCGGTGAGT 223 181a_2 miRNA-043 mir-213/mir- 1388 let-7f (Michael ACCATCGACCGTTGATTGTACC 253 181a_2 et al) hypothetical 1389 mir-26a TAGGCCAAATGGCGCATCAAT 1124 miRNA 090 mir-138_2 1390 mir-92 AGCTGGTGTTGTGAATC 256 mir-138_2 1390 miR-27* (Michael AGCTGGTGTTGTGAATCAGGCCG 1127 et al) mir-196_1 1391 miR-29b (RFAM- TAGGTAGTTTCATGTTGTTGG 1097 Human) mir-196_1 1391 mir-7 TAGGTAGTTTCATGTTGTTGGG 228 mir-199a_2 1392 miR-202 (mouse) CCCAGTGTTCAGACTACCTGTT 1128 mir-199a_2 1392 mir-15a CCCAGTGTTCAGACTACCTGTTC 259 mir-199a_2 1392 mir-211 (rodent) TACAGTAGTCTGCACATTGGTT 1118 mir-181b_1 1393 mir-16 AACATTCATTGCTGTCGGTGGGTT 260 hypothetical 1394 miR-26a (Michael TGACAGTCAATTAACAAGTTT 1130 miRNA 101 et al) hypothetical 1395 mir-127_Ruvkun TTCCTCCTCCTCCGACTCGGA 1135 miRNA 111 mir-218_2 1396 mir-33a TTGTGCTTGATCTAACCATGT 234 mir-218_2 1396 mir-24 TTGTGCTTGATCTAACCATGTG 1103 mir-148b 1397 mir-30d TCAGTGCATCACAGAACTTTGT 272 mir-216 1398 mir-30d_Ruvkun TAATCTCAGCTGGCAACTGTG 274 hypothetical 1399 miR-136 TAAACTGGCTGATAATTTTTG 1141 miRNA 137 hypothetical 1400 miR-154 TGCAAGTATGAAAATGAGATT 1142 miRNA 138 mir-210 1401 let-7c CTGTGCGTGTGACAGCGGCTG 277 mir-223 1402 let-7c_Ruvkun TGTCAGTTTGTCAAATACCCC 279 hypothetical 1403 miR-149 TGCAAGCAGATGCTGATAATA 1145 miRNA 153 hypothetical 1404 mir-30c TTAAAGTGGATGTGTGTTATT 1146 miRNA 154 mir-135_1 1405 hypothetical TATGGCTTTTTATTCCTATGTGA 1149 miRNA-101 mir-135_1 1405 let-7e TATGGCTTTTTATTCCTATGTGAT 283 non-coding RNA in 1406 mir-181b TATGGCTTTTTATTCCTATGTGA 1149 rhabdomyosarcoma/ mir-135_2 non-coding RNA in 1406 miR-155/ TATGGCTTTTTATTCCTATGTGAT 283 rhabdomyosarcoma/ hypothetical mir-135_2 miRNA-071 hypothetical 1407 mir-30c_Ruvkun TGATCTTGCTCTAACACTTGG 1157 miRNA 170 glutamate 174 miR-99b TGACAAGTATGTTTTATCGTT 1158 receptor, ionotropic, AMPA 2/hypothetical miRNA-171 hypothetical 179 miR-125a TAGGAGTTTGATATGACATAT 1163 miRNA 176 hypothetical 1408 mir-125b TGAAAGGCACTTTGTCCAATT 1166 miRNA 179 hypothetical 1409 mir-221 TCACCTGCTCTGGAAGTAGTT 1167 miRNA 181 mir-181c 1410 mir-133a AACATTCAACCTGTCGGTGAGT 290 mir-100_1 1411 let-7b AACCCGTAGATCCGAACTTGTG 275 mir-103_1 950 mir-29a AGCAGCATTGTACAGGGCTATGA 225 mir-107 1412 mir-141 AGCAGCATTGTACAGGGCTATCA 229 mir-19a 1413 mir-20 TGTGCAAATCTATGCAAAACTGA 268 mir-19b_1 1414 mir-21 AGTTTTGCAGGTTTGCATCCAGC 1179 mir-19b_1 1414 mir-223 TGTGCAAATCCATGCAAAACTGA 241 mir-92_1 1415 hypothetical TATTGCACTTGTCCCGGCCTG 1182 miRNA-090 mir-92_1 1415 miR-9 TATTGCACTTGTCCCGGCCTGT 216 mir-98 1416 mir-131 TGAGGTAGTAAGTTGTATTGTT 257 mir-104 1417 mir-221 (RFAM- TCAACATCAGTCTGATAAGCTA 335 (Mourelatos) mmu) mir-27 1418 mir-213 TTCACAGTGGCTAAGTTCC 1186 (Mourelatos) mir-27 1418 mir-222 (RFAM- TTCACAGTGGCTAAGTTCCGC 1187 (Mourelatos) mmu) mir-27 1418 mir-203 TTCACAGTGGCTAAGTTCCGCC 1188 (Mourelatos) mir-31 1419 mir-178 (Kosik) AGGCAAGATGCTGGCATAGCTG 1197 mir-31 1419 miR-203 (RFAM- GGCAAGATGCTGGCATAGCTG 1198 M. mu.) mir-32 1420 let-7g TATTGCACATTACTAAGTTGC 1199 mir_186 1421 miR-326 (Ruvkun) CAAAGAATTCTCCTTTTGGGCTT 1208 mir_191 1422 mir-329 (mouse) CAACGGAATCCCAAAAGCAGCT 1210 mir_191 1422 miR-27a (RFAM- CAACGGAATCCCAAAAGCAGCTGT 1211 Human) mir_195 1423 mir-330 (rodent) TAGCAGCACAGAAATATTGGC 1216 mir_193 1424 mir-337 (rodent) AACTGGCCTACAAAGTCCCAG 1217 mir_188 1425 mir-345 (rodent) CATCCCTTGCATGGTGGAGGGT 1219 mir_208 1426 mir-346 (mouse) ATAAGACGAGCAAAAAGCTTGT 1222 mir_139 1427 mir-151* (Ruvkun) TCTACAGTGCACGTGTCT 1223 mir-200b 1428 mir-151 (rodent) CTCTAATACTGCCTGGTAATGATG 1224 mir-200b 1428 mir-216 TAATACTGCCTGGTAATGATGA 1225 mir-200b 1428 mir-219 TAATACTGCCTGGTAATGATGAC 1226 mir-200a 1429 mir-181a TAACACTGTCTGGTAACGATG 1227 mir-200a 1429 mir-151L (rodent) TAACACTGTCTGGTAACGATGT 1228 mir-227* 1430 mir-191 ACTGCCCCAGGTGCTGCTGG 1231 (Kosik)/mir-226* (Kosik) mir-227* 1430 hypothetical CCACTGCCCCAGGTGCTGCTGG 1232 (Kosik)/mir-226* miRNA-058 (Kosik) mir-227* 1430 hypothetical CGCATCCCCTAGGGCATTGGTGT 1233 (Kosik)/mir-226* miRNA-055 (Kosik) mir-244* (Kosik) 1431 mir-218 TCCAGCATCAGTGATTTTGTTGA 1234 mir-224* (Kosik) 1432 mir-253* (Kosik) GCACATTACACGGTCGACCTCT 1235 mir-248* (Kosik) 1433 mir-222 TCTCACACAGAAATCGCACCCGTC 1236 mir-138_3 1434 mir-19b* (Michael AGCTGGTGTTGTGAATC 256 et al) mir-138_3 1434 mir-27b AGCTGGTGTTGTGAATCAGGCCG 1127 mir-181b_2 1435 mir-15_Ruvkun AACATTCATTGCTGTCGGTGGGTT 260 mir-103_2 1436 miR-101 (RFAM- AGCAGCATTGTACAGGGCTATGA 225 Human) mir-134 (Sanger) 1437 mir-129 TGTGACTGGTTGACCAGAGGG 1240 mir-146 (Sanger) 1438 mir-129as/mir- TGAGAACTGAATTCCATGGGTT 1241 258* (Kosik) mir-30e 1439 miR-129b (RFAM- TGTAAACATCCTTGACTGGA 1243 (RFAM/mmu) Human) mir-30e 1439 miR-135 (RFAM- TGTAAACATCCTTGACTGGAAG 1244 (RFAM/mmu) Human) mir-299 1440 mir-133b TGGTTTACCGTCCCACATACAT 1246 (RFAM/mmu) mir-340 (Ruvkun) 1441 miR-188 TCCGTCTCAGTTACTTTATAGCC 1257 mir-331 (Ruvkun) 1442 miR-208 GCCCCTGGGCCTATCCTAGAA 1258 mir-187 1443 miR-199-s TCGTGTCTTGTGTTGCAGCCG 1270 mir-187 1443 let-7b_Ruvkun TCGTGTCTTGTGTTGCAGCCGG 276 miR-201 1444 miR-187 (RFAM- TACTCAGTAAGGCATTGTTCT 1479 Human) miR-207 1445 miR-201 GCTTCTCCTGGCTCTCCTCCCTC 1480 miR-291 1446 miR-291 AAAGTGCTTCCACTTTGTGTGCC 1481 miR-291 1446 miR-207 CATCAAAGTGGAGGCCCTCTCT 1482 miR-292 1447 miR-291 AAGTGCCGCCAGGTTTTGAGTGT 1483 miR-292 1447 miR-292 ACTCAAACTGGGGGCTCTTTTG 1484 miR-293 1448 miR-292 AGTGCCGCAGAGTTTGTAGTGT 1485 miR-294 1449 miR-293 AAAGTGCTTCCCTTTTGTGTGT 1486 miR-295 1450 miR-294 AAAGTGCTACTACTTTTGAGTCT 1487 miR-300 1451 miR-295 TATGCAAGGGCAAGCTCTCTTC 1488 miR-322 1452 miR-300 AAACATGAAGCGCTGCAACA 1489 miR-344 1453 miR-322 TGATCTAGCCAAAGCCTGACTGT 1490 miR-350 1454 miR-344 TTCACAAAGCCCATACACTTTCAC 1491 miR-290 1455 miR-350 CTCAAACTATGGGGGCACTTTTT 1492 miR-351 1456 miR-290 TCCCTGAGGAGCCCTTTGAGCCTG 1493 miR-341 1457 miR-351 TCGATCGGTCGGTCGGTCAGT 1494 miR-298 1458 miR-341 GGCAGAGGAGGGCTGTTCTTCC 1495

A list of rat pri-miRNAs and the mature miRNAs predicted to derive from them is shown in Table 62. “Pri-miRNA name” indicates the gene name for each of the pri-miRNAs. Also given in table 62 are the name and sequence of the mature miRNA derived from the pri-miRNA. Mature miRNA sequences from pri-miRNA precursors have been proposed by several groups; consequently, for a given pri-miRNA sequence, several miRNAs may be disclosed and given unique names, and thus a given pri-miRNA sequence may occur repeatedly in the table. The sequences are written in the 5′ to 3′ direction and are represented in the DNA form. It is understood that a person having ordinary skill in the art would be able to convert the sequence of the targets to their RNA form by simply replacing the thymidine (T) with uracil (U) in the sequence.

TABLE 62 Rat pri-miRNA sequences and the corresponding mature miRNAs SEQ SEQ ID ID Pri-miRNA name NO Mature miRNA name Mature miRNA sequence NO mir-20 1496 miR-20* (mouse) ACTGCATTACGAGCACTTACA 1608 mir-20 1496 miR-20 (RFAM- TAAAGTGCTTATAGTGCAGGTA 1126 Human) mir-20 1496 mir-20 TAAAGTGCTTATAGTGCAGGTAG 254 mir-151* (Ruvkun) 1497 mir-151L (rodent) ACTAGACTGAGGCTCCTTGAGG 1476 mir-151* (Ruvkun) 1497 mir-151 (rodent) CTAGACTGAGGCTCCTTGAGG 1477 mir-151* (Ruvkun) 1497 mir-151* (Ruvkun) TCGAGGAGCTCACAGTCTAGTA 1256 mir-346 (Ruvkun) 1498 miR-346 (rat) TGTCTGCCTGAGTGCCTGCCTCT 1609 mir-143 1499 miR-143 (Michael TGAGATGAAGCACTGTAGCTC 1088 et al) mir-143 1499 mir-143 TGAGATGAAGCACTGTAGCTCA 220 mir-203 1500 mir-203 GTGAAATGTTTAGGACCACTAG 197 mir-203 1500 miR-203 (RFAM-M. mu.) TGAAATGTTTAGGACCACTAG 1068 mir-203 1500 miR-203 (Tuschl) TGAAATGTTTAGGACCACTAGA 1069 mir-26b 1501 miR-26b (RFAM- TTCAAGTAATTCAGGATAGGT 1147 Human) mir-26b 1501 mir-26b TTCAAGTAATTCAGGATAGGTT 281 mir-128a 1276 mir-128 (Kosik) TCACAGTGAACCGGTCTCTTT 1073 mir-128a 1276 mir-128a TCACAGTGAACCGGTCTCTTTT 200 mir-29b_1 1277 miR-29b (RFAM- TAGCACCATTTGAAATCAGT 1172 Human) mir-29b_1 1277 miR-29b (RFAM-M. mu.) TAGCACCATTTGAAATCAGTGT 1173 mir-29b_1 1502 mir-29b TAGCACCATTTGAAATCAGTGTT 195 mir-29c 1278 mir-29c CTAGCACCATTTGAAATCGGTT 232 mir-29c 1278 miR-29c (Tuschl) TAGCACCATTTGAAATCGGTTA 1100 mir-123/mir-126 1503 mir-123/mir-126as CATTATTACTTTTGGTACGCG 205 mir-123/mir-126 1503 mir-126 TCGTACCGTGAGTAATAATGC 1076 mir-130a 1504 mir-130a CAGTGCAATGTTAAAAGGGC 233 mir-130a 1504 mir-130 (Kosik) CAGTGCAATGTTAAAAGGGCAT 1101 mir-124a_3 1282 mir-124a (Kosik) TAAGGCACGCGGTGAATGCCA 1104 mir-124a_3 1282 mir-124a TTAAGGCACGCGGTGAATGCCA 235 mir-124a_3 1282 mir-124a_Ruvkun TTAAGGCACGCGGTGAATGCCAA 1105 mir-15b 1286 miR-15b (Michael TAGCAGCACATCATGGTTTAC 1115 et al) mir-15b 1286 mir-15b TAGCAGCACATCATGGTTTACA 246 mir-16_3 1505 mir-16 TAGCAGCACGTAAATATTGGCG 196 mir-16_3 1505 mir-16_Ruvkun TAGCAGCACGTAAATATTGGCGT 1176 mir-137 1288 mir-137 TATTGCTTAAGAATACGCGTAG 270 mir-101_1 1289 mir-101 TACAGTACTGTGATAACTGA 265 mir-101_1 1289 miR-101 (RFAM- TACAGTACTGTGATAACTGAAG 1170 Human) mir-29a 1291 mir-29a CTAGCACCATCTGAAATCGGTT 247 mir-29a 1291 mir-29a_Ruvkun TAGCACCATCTGAAATCGGTTA 1116 mir-29b_2 1292 miR-29b (RFAM- TAGCACCATTTGAAATCAGT 1172 Human) mir-29b_2 1292 miR-29b (RFAM-M. mu.) TAGCACCATTTGAAATCAGTGT 1173 mir-29b_2 1292 mir-29b TAGCACCATTTGAAATCAGTGTT 195 mir-131_3/mir-9 1506 mir-131 TAAAGCTAGATAACCGAAAGT 211 mir-131_3/mir-9 1506 mir-131_Ruvkun TAAAGCTAGATAACCGAAAGTA 1080 mir-131_3/mir-9 1506 miR-9 TCTTTGGTTATCTAGCTGTATGA 1081 mir-23a 1507 mir-23a ATCACATTGCCAGGGATTTCC 289 mir-140 1508 mir-140 AGTGGTTTTACCCTATGGTAG 192 mir-140 1508 miR-140-as TACCACAGGGTAGAACCACGGA 1065 mir-140 1508 mir-239* (Kosik) TACCACAGGGTAGAACCACGGACA 1066 mir-125b_1 1509 mir-125b TCCCTGAGACCCTAACTTGTGA 258 mir-26a_1 1510 miR-26a (Michael TTCAAGTAATCCAGGATAGGC 1203 et al) mir-26a_1 1510 mir-26a TTCAAGTAATCCAGGATAGGCT 226 let-7i 1302 let-7i TGAGGTAGTAGTTTGTGCT 209 let-7i 1302 let-7i_Ruvkun TGAGGTAGTAGTTTGTGCTGTT 1078 mir-21 1511 mir-21 TAGCTTATCAGACTGATGTTGA 236 mir-22 1512 mir-22 AAGCTGCCAGTTGAAGAACTGT 215 mir-142 1513 mir-142 CATAAAGTAGAAAGCACTAC 217 mir-142 1513 miR-142-as TGTAGTGTTTCCTACTTTATGG 1086 mir-142 1513 miR-142as TGTAGTGTTTCCTACTTTATGGA 1087 (Michael et al) mir-144 1514 mir-144 TACAGTATAGATGATGTACTAG 237 mir-152 1515 mir-152 TCAGTGCATGACAGAACTTGG 282 mir-153_2 1516 mir-153 TTGCATAGTCACAAAAGTGA 201 let-7a_1 1517 let-7a TGAGGTAGTAGGTTGTATAGTT 222 let-7d 1518 let-7d AGAGGTAGTAGGTTGCATAGT 245 let-7d 1518 let-7d_Ruvkun AGAGGTAGTAGGTTGCATAGTT 1113 let-7d 1518 let-7d* (RFAM-M. mu.) CTATACGACCTGCTGCCTTTCT 1114 let-7f_1 1519 let-7f (Michael TGAGGTAGTAGATTGTATAGT 1098 et al) let-7f_1 1519 let-7f TGAGGTAGTAGATTGTATAGTT 231 miR-24-1 1313 miR-189 (RFAM- GTGCCTACTGAGCTGATATCAGT 1271 Human) miR-24-1 1313 mir-24 TGGCTCAGTTCAGCAGGAACAG 264 mir-124a_1 1318 mir-124a (Kosik) TAAGGCACGCGGTGAATGCCA 1104 mir-124a_1 1318 mir-124a TTAAGGCACGCGGTGAATGCCA 235 mir-124a_1 1318 mir-124a_Ruvkun TTAAGGCACGCGGTGAATGCCAA 1105 mir-18 1319 mir-18 TAAGGTGCATCTAGTGCAGATA 262 mir-18 1319 mir-18_Ruvkun TAAGGTGCATCTAGTGCAGATAG 1177 mir-30b 1520 mir-30b TGTAAACATCCTACACTCAGC 266 mir-30b 1520 mir-30b_Ruvkun TGTAAACATCCTACACTCAGCT 1137 mir-30d 1521 mir-30d TGTAAACATCCCCGACTGGAAG 240 mir-30d 1521 mir-30d_Ruvkun TGTAAACATCCCCGACTGGAAGCT 1108 let-7b 1522 let-7b TGAGGTAGTAGGTTGTGTGGTT 212 let-7b 1522 let-7b_Ruvkun TGAGGTAGTAGGTTGTGTGGTTT 1082 let-7e 1328 let-7e TGAGGTAGGAGGTTGTATAGT 249 mir-133a_1 1330 mir-133a TTGGTCCCCTTCAACCAGCTGT 255 mir-145 1332 miR-145 (Michael GTCCAGTTTTCCCAGGAATCC 1122 et al) mir-145 1332 mir-145 GTCCAGTTTTCCCAGGAATCCCTT 252 mir-122a 1523 miR-122a,b TGGAGTGTGACAATGGTGTTTG 1084 (Tuschl) mir-122a 1523 mir-122a TGGAGTGTGACAATGGTGTTTGT 214 let-7f_2 1335 let-7f (Michael TGAGGTAGTAGATTGTATAGT 1098 et al) let-7f_2 1335 let-7f TGAGGTAGTAGATTGTATAGTT 231 mir-127 1337 mir-127_Ruvkun TCGGATCCGTCTGAGCTTGG 1204 mir-127 1337 miR-127 TCGGATCCGTCTGAGCTTGGCT 1205 mir-136 1338 miR-136 ACTCCATTTGTTTTGATGATGGA 1206 mir-154 1339 miR-154 TAGGTTATCCGTGTTGCCTTCG 1207 mir-30c_2 1341 mir-30c TGTAAACATCCTACACTCTCAGC 280 mir-30c_2 1341 mir-30c_Ruvkun TGTAAACATCCTACACTCTCAGCT 1129 mir-99b 1342 miR-99b CACCCGTAGAACCGACCTTGCG 1201 MiR-125a 1524 miR-125a TCCCTGAGACCCTTTAACCTGTG 1202 mir-221 1525 mir-221 (RFAM- AGCTACATTGTCTGCTGGGTTT 1106 mmu) mir-221 1525 mir-221 AGCTACATTGTCTGCTGGGTTTC 238 mir-101_3 1526 mir-101b (rodent) TACAGTACTGTGATAGCTGAAG 1460 mir-17/mir-91 1527 mir-17 (human, ACTGCAGTGAAGGCACTTGT 1180 rat) mir-17/mir-91 1527 mir-91_Ruvkun CAAAGTGCTTACAGTGCAGGTAG 1181 mir-17/mir-91 1527 mir-17as/mir-91 CAAAGTGCTTACAGTGCAGGTAGT 204 hypothetical 1528 hypothetical TTCCTATGCATATACTTCTTT 1132 miRNA 105 miRNA-105 mir-211 1529 mir-211 (rodent) TTCCCTTTGTCATCCTTTGCCT 1465 mir-217 1530 mir-217 (rodent) TACTGCATCAGGAACTGACTGGAT 1466 mir-7_3 1531 mir-7b (rodent) TGGAAGACTTGTGATTTTGTT 1468 mir-325 (Ruvkun) 1357 mir-325 (rodent) CCTAGTAGGTGCTCAGTAAGTGT 1469 mir-326 (Ruvkun) 1532 miR-326 (Ruvkun) CCTCTGGGCCCTTCCTCCAG 1263 mir-326 (Ruvkun) 1532 mir-326 (rodent) CCTCTGGGCCCTTCCTCCAGT 1470 mir-330 (Ruvkun) 1533 mir-330 (rodent) GCAAAGCACAGGGCCTGCAGAGA 1472 mir-337 (Ruvkun) 1361 mir-337 (rodent) TTCAGCTCCTATATGATGCCTTT 1473 mir-345 (Ruvkun) 1362 mir-345 (rodent) TGCTGACCCCTAGTCCAGTGC 1474 mir_34b (RFAM) 1365 mir-34b (mouse) TAGGCAGTGTAATTAGCTGATTG 1478 mir-34 1534 mir-34 TGGCAGTGTCTTAGCTGGTTGT 194 mir-34 1534 miR-172 (RFAM-M. mu.) TGGCAGTGTCTTAGCTGGTTGTT 1067 mir-7_1/mir-7_1* 1535 mir-7_1*_Ruvkun CAACAAATCACAGTCTGCCATA 1070 mir-7_1/mir-7_1* 1535 mir-7 TGGAAGACTAGTGATTTTGTT 198 mir-10b 1536 miR-10b (Tuschl) CCCTGTAGAACCGAATTTGTGT 1071 mir-10b 1536 mir-10b TACCCTGTAGAACCGAATTTGT 199 mir-10b 1536 miR-10b (Michael TACCCTGTAGAACCGAATTTGTG 1072 et al) mir-132 1370 miR-132 (RFAM- TAACAGTCTACAGCCATGGTCG 1077 Human) mir-132 1370 mir-132 TAACAGTCTACAGCCATGGTCGC 206 mir-212 1537 mir-212 TAACAGTCTCCAGTCACGGCC 210 mir-108_1 1538 mir-108 ATAAGGATTTTTAGGGGCATT 207 hypothetical 26 hypothetical TGGGCAAGAGGACTTTTTAAT 1079 miRNA 023 miRNA-023 mir-214 1539 mir-214 ACAGCAGGCACAGACAGGCAG 219 hypothetical 43 hypothetical TGTCAACAAAACTGCTTACAA 1092 miRNA 040 miRNA-040 hypothetical 1540 hypothetical TGACAGGAAATCTTTGAGAGG 1094 miRNA 043 miRNA-043 mir-205 1541 mir-205 TCCTTCATTCCACCGGAGTCTG 224 mir-33a 1542 mir-33a GTGCATTGTAGTTGCATTG 227 mir-196_2 1543 miR-196 (Tuschl) TAGGTAGTTTCATGTTGTTGG 1097 mir-196_2 1543 mir-196 TAGGTAGTTTCATGTTGTTGGG 228 mir-218_1 1544 mir-218 TTGTGCTTGATCTAACCATGT 234 mir-218_1 1544 mir-253* (Kosik) TTGTGCTTGATCTAACCATGTG 1103 mir-222 1545 mir-222 (RFAM- AGCTACATCTGGCTACTGGGTCT 1107 mmu) mir-222 1545 mir-222 AGCTACATCTGGCTACTGGGTCTC 239 mir-128b 1381 mir-128 (Kosik) TCACAGTGAACCGGTCTCTTT 1073 mir-128b 1381 mir-128b TCACAGTGAACCGGTCTCTTTC 242 mir-219_2 1546 mir-219 TGATTGTCCAAACGCAATTCT 271 hypothetical 1547 hypothetical TCACATTTGCCTGCAGAGATT 1109 miRNA 070 miRNA-070 mir-129_2 1548 mir-129as/mir- AAGCCCTTACCCCAAAAAGCAT 1110 258* (Kosik) mir-129_2 1548 mir-129 CTTTTTGCGGTCTGGGCTTGC 243 mir-129_2 1548 miR-129b (RFAM- CTTTTTGCGGTCTGGGCTTGCT 1111 Human) mir-133b 1385 mir-133b TTGGTCCCCTTCAACCAGCTA 244 hypothetical 78 hypothetical TGGTTAAAATATTAATGGGGC 1112 miRNA 075 miRNA-075 mir-204 1549 mir-204 TTCCCTTTGTCATCCTATGCCT 251 mir-204 1549 miR-204 (Tuschl) TTCCCTTTGTCATCCTATGCCTG 1121 mir-213/mir- 1550 mir-178 (Kosik) AACATTCAACGCTGTCGGTGAG 1096 181a_2 mir-213/mir- 1550 mir-181a AACATTCAACGCTGTCGGTGAGT 223 181a_2 mir-213/mir- 1550 mir-213 ACCATCGACCGTTGATTGTACC 253 181a_2 hypothetical 1551 hypothetical TAGGCCAAATGGCGCATCAAT 1124 miRNA 090 miRNA-090 mir-138_2 1552 mir-138 AGCTGGTGTTGTGAATC 256 mir-138_2 1552 mir-138_Ruvkun AGCTGGTGTTGTGAATCAGGCCG 1127 mir-199a_2 1553 miR-199-s CCCAGTGTTCAGACTACCTGTT 1128 mir-199a_2 1553 mir-199a CCCAGTGTTCAGACTACCTGTTC 259 mir-199a_2 1553 miR-199-as TACAGTAGTCTGCACATTGGTT 1118 hypothetical 1554 hypothetical TGACAGTCAATTAACAAGTTT 1130 miRNA 101 miRNA-101 mir-148b 1397 mir-148b TCAGTGCATCACAGAACTTTGT 272 mir-216 1555 mir-216 TAATCTCAGCTGGCAACTGTG 274 hypothetical 1399 hypothetical TAAACTGGCTGATAATTTTTG 1141 miRNA 137 miRNA-137 hypothetical 1556 hypothetical TGCAAGTATGAAAATGAGATT 1142 miRNA 138 miRNA-138 mir-210 1557 mir-210 CTGTGCGTGTGACAGCGGCTG 277 mir-223 1558 mir-223 TGTCAGTTTGTCAAATACCCC 279 hypothetical 1404 hypothetical TTAAAGTGGATGTGTGTTATT 1146 miRNA 154 miRNA-154 non-coding RNA in 13 miR-135 (RFAM- TATGGCTTTTTATTCCTATGTGA 1149 rhabdomyosarcoma/ Human) mir-135_2 non-coding RNA in 13 mir-135 TATGGCTTTTTATTCCTATGTGAT 283 rhabdomyosarcoma/ mir-135_2 hypothetical 1559 hypothetical TGATCTTGCTCTAACACTTGG 1157 miRNA 170 miRNA-170 glutamate 174 hypothetical TGACAAGTATGTTTTATCGTT 1158 receptor, miRNA-171 ionotropic, AMPA 2/hypothetical miRNA-171 hypothetical 179 hypothetical TAGGAGTTTGATATGACATAT 1163 miRNA 176 miRNA-176 hypothetical 1560 hypothetical TGAAAGGCACTTTGTCCAATT 1166 miRNA 179 miRNA-179 hypothetical 1409 hypothetical TCACCTGCTCTGGAAGTAGTT 1167 miRNA 181 miRNA-181 mir-181c 1410 mir-181c AACATTCAACCTGTCGGTGAGT 290 mir-100_1 1561 mir-100 AACCCGTAGATCCGAACTTGTG 275 mir-103_1 950 mir-103 AGCAGCATTGTACAGGGCTATGA 225 mir-107 1562 mir-107 AGCAGCATTGTACAGGGCTATCA 229 mir-19a 1563 mir-19a TGTGCAAATCTATGCAAAACTGA 268 mir-19b_1 1414 mir-19b* (Michael AGTTTTGCAGGTTTGCATCCAGC 1179 et al) mir-19b_1 1414 mir-19b TGTGCAAATCCATGCAAAACTGA 241 mir-92_1 1564 miR-92 (RFAM-M. mu.) TATTGCACTTGTCCCGGCCTG 1182 mir-92_1 1564 mir-92 TATTGCACTTGTCCCGGCCTGT 216 mir-98 1565 mir-98 TGAGGTAGTAAGTTGTATTGTT 257 mir-104 1566 miR-104 TCAACATCAGTCTGATAAGCTA 335 (Mourelatos) (Mourelatos) mir-27 1567 miR-27 TTCACAGTGGCTAAGTTCC 1186 (Mourelatos) (Mourelatos) mir-27 1567 miR-27a (RFAM-M. mu.) TTCACAGTGGCTAAGTTCCGC 1187 (Mourelatos) mir-27 1567 miR-27a (RFAM- TTCACAGTGGCTAAGTTCCGCC 1188 (Mourelatos) Human) mir-31 1568 miR-31 (RFAM-M. mu.) AGGCAAGATGCTGGCATAGCTG 1197 mir-31 1568 miR-31 (Tuschl) GGCAAGATGCTGGCATAGCTG 1198 mir-32 1569 miR-32 (Tuschl) TATTGCACATTACTAAGTTGC 1199 mir_186 1570 miR-186 CAAAGAATTCTCCTTTTGGGCTT 1208 mir_191 1571 mir-191 CAACGGAATCCCAAAAGCAGCT 1210 mir_191 1422 mir-191_Ruvkun CAACGGAATCCCAAAAGCAGCTGT 1211 mir_195 1572 miR-195 TAGCAGCACAGAAATATTGGC 1216 mir_193 1573 miR-193 AACTGGCCTACAAAGTCCCAG 1217 mir_208 1574 miR-208 ATAAGACGAGCAAAAAGCTTGT 1222 mir_139 1427 miR-139 TCTACAGTGCACGTGTCT 1223 mir-200b 1428 miR-200a (RFAM- CTCTAATACTGCCTGGTAATGATG 1224 Human) mir-200b 1428 miR-200b (Michael TAATACTGCCTGGTAATGATGA 1225 et al) mir-200b 1428 miR-200b TAATACTGCCTGGTAATGATGAC 1226 mir-200a 1429 miR-200a TAACACTGTCTGGTAACGATG 1227 mir-200a 1429 miR-200a (RFAM-M. mu.) TAACACTGTCTGGTAACGATGT 1228 mir-227* 1430 mir-226* (Kosik) ACTGCCCCAGGTGCTGCTGG 1231 (Kosik)/mir-226* (Kosik) mir-227* 1430 mir-324-3p_Ruvkun CCACTGCCCCAGGTGCTGCTGG 1232 (Kosik)/mir-226* (Kosik) mir-227* 1430 mir-227* (Kosik) CGCATCCCCTAGGGCATTGGTGT 1233 (Kosik)/mir-226* (Kosik) mir-244* (Kosik) 1431 mir-244* (Kosik) TCCAGCATCAGTGATTTTGTTGA 1234 mir-224* (Kosik) 1432 mir-224* (Kosik) GCACATTACACGGTCGACCTCT 1235 mir-248* (Kosik) 1433 mir-248* (Kosik) TCTCACACAGAAATCGCACCCGTC 1236 mir-138_3 1575 mir-138 AGCTGGTGTTGTGAATC 256 mir-138_3 1575 mir-138_Ruvkun AGCTGGTGTTGTGAATCAGGCCG 1127 mir-181b_2 1576 mir-181b AACATTCATTGCTGTCGGTGGGTT 260 mir-134 (Sanger) 1289 miR-134 (RFAM- TGTGACTGGTTGACCAGAGGG 1240 Human) mir-146 (Sanger) 1577 miR-146 (RFAM- TGAGAACTGAATTCCATGGGTT 1241 Human) mir-30e 1578 miR-30e (RFAM-M. mu.) TGTAAACATCCTTGACTGGA 1243 (RFAM/mmu) mir-30e 1578 miR-97 (Michael TGTAAACATCCTTGACTGGAAG 1244 (RFAM/mmu) et al) mir-299 1440 miR-299 (RFAM-M. mu.) TGGTTTACCGTCCCACATACAT 1246 (RFAM/mmu) mir-34a 1579 mir-34c (RFAM) AGGCAGTGTAGTTAGCTGATTG 1250 (RFAM/mmu) mir-34a 1579 miR-34a (RFAM-M. mu.) AGGCAGTGTAGTTAGCTGATTGC 1251 (RFAM/mmu) mir-135b (Ruvkun) 1580 mir-135b (Ruvkun) TATGGCTTTTCATTCCTATGTG 1254 mir-331 (Ruvkun) 1442 mir-331 (Ruvkun) GCCCCTGGGCCTATCCTAGAA 1258 mir-187 1443 miR-187 (RFAM- TCGTGTCTTGTGTTGCAGCCG 1270 Human) mir-187 1443 mir-187 TCGTGTCTTGTGTTGCAGCCGG 276 collagen, type I, 1581 hypothetical AGACATGTTCAGCTTTGTGGA 1063 alpha 1/ miRNA-144 hypothetical miRNA-144 DiGeorge syndrome 1582 hypothetical TGTGATTTCCAATAATTGAGG 1123 critical region miRNA-088 gene 8/ hypothetical miRNA-088 hypothetical miR- 1583 miR-190 TGATATGTTTGATATATTAGGT 1075 13/miR-190 hypothetical 1584 hypothetical TAAGACTTGCAGTGATGTTTA 1091 miRNA 039 miRNA-039 hypothetical 1585 hypothetical TACCAGTTGTTTTCTCTGTGA 1093 miRNA 041 miRNA-041 hypothetical 47 hypothetical TTCCACTCTGTTTATCTGACA 1095 miRNA 044 miRNA-044 hypothetical 86 hypothetical TTACATGGGGAAGCTATCATA 1119 miRNA 083 miRNA-083 hypothetical 1586 hypothetical TGACAGTTTATTGGCTTTATC 1133 miRNA 107 miRNA-107 mir-10a 1587 mir-10a (Tuschl) TACCCTGTAGATCCGAATTTGT 1139 mir-10a 1587 mir-10a TACCCTGTAGATCCGAATTTGTG 267 mir-130b 1588 mir-130b CAGTGCAATGATGAAAGGGC 273 mir-130b 1588 mir-266* (Kosik) CAGTGCAATGATGAAAGGGCAT 1140 hypothetical 1589 hypothetical AGACAAACATGCTACTCTCAC 1164 miRNA-177_1 miRNA-177 mir_185 1590 miR-185 TGGAGAGAAAGGCAGTTC 1218 mir_194_2 1591 miR-194 TGTAACAGCAACTCCATGTGGA 1221 mir-150 (Sanger) 1592 miR-150 (RFAM- TCTCCCAACCCTTGTACCAGTG 1242 Human) mir-301 1593 miR-301 (RFAM-M. mu.) CAGTGCAATAGTATTGTCAAAGC 1247 (RFAM/mmu) mir-301 1593 mir-301_Ruvkun CAGTGCAATAGTATTGTCAAAGCAT 1248 (RFAM/mmu) mir_320 1594 miR-320 AAAAGCTGGGTTGAGAGGGCGAA 1252 mir_200c (RFAM) 1595 mir-200c (RFAM) AATACTGCCGGGTAATGATGGA 1259 miR-322 1596 miR-322 AAACATGAAGCGCTGCAACA 1489 miR-341 1457 miR-341 TCGATCGGTCGGTCGGTCAGT 1494 miR-344 1597 miR-344 TGATCTAGCCAAAGCCTGACCGT 1610 miR-350 1598 miR-350 TTCACAAAGCCCATACACTTTCAC 1491 miR-351 1599 miR-351 TCCCTGAGGAGCCCTTTGAGCCTG 1493 miR-290 1600 miR-290 CTCAAACTATGGGGGCACTTTTT 1492 miR-291 1601 miR-291 AAAGTGCTTCCACTTTGTGTGCC 1481 miR-291 1601 miR-291 CATCAAAGTGGAGGCCCTCTCT 1482 miR-292 1602 miR-292 AAGTGCCGCCAGGTTTTGAGTGT 1483 miR-292 1602 miR-292 ACTCAAACTGGGGGCTCTTTTG 1484 miR-298 1603 miR-298 GGCAGAGGAGGGCTGTTCTTCC 1495 miR-300 1604 miR-300 TATGCAAGGGCAAGCTCTCTTC 1488 miR-333 1605 miR-333 GTGGTGTGCTAGTTACTTTT 1611 miR-336 1606 miR-336 TCACCCTTCCATATCTAGTCT 1612 miR-349 1607 miR-349 CAGCCCTGCTGTCTTAACCTCT 1613

A list of Drosophila pri-miRNAs and the mature miRNAs predicted to derive from them is shown in Table 63. “Pri-miRNA name” indicates the gene name for each of the pri-miRNAs, and “pri-miRNA sequence” indicates the sequence of the predicted primary miRNA transcript. Also given in table 63 are the name and sequence of the mature miRNA derived from the pri-miRNA. The sequences are written in the 5′ to 3′ direction and are represented in the DNA form. It is understood that a person having ordinary skill in the art would be able to convert the sequence of the targets to their RNA form by simply replacing the thymidine (T) with uracil (U) in the sequence.

TABLE 63 Drosophila pri-miRNA sequences and the corresponding mature miRNAs Pri- SEQ SEQ miRNA ID Mature miRNA ID name Pri-miRNA sequence NO name Mature miRNA sequence NO mir-14 GGAGCGAGACGGGGACTCACT 1614 miR-14 TCAGTCTTTTTCTCTCTCCTA 1616 GTGCTTATTAAATAGTCAGTC TTTTTCTCTCTCCTATACAAA TTGCGGGC mir- AATGATTTGACTACGAAACCG 1615 mir-Bantam GTGAGATCATTTTGAAAGCTG 1617 bantam GTTTTCGATTTGGTTTGACTG TTTTTCATACAAGTGAGATCA TTTTGAAAGCTGATTTTGTCA ATGAATA

Oligomeric compounds targeting or mimicking pri-miRNAs, pre-miRNAs, or miRNAs were given internal numerical identifiers (ISIS Numbers) and are shown in Tables 64, 65, and 66 respectively. The sequences are written in the 5′ to 3′ direction and are represented in the DNA form. It is understood that a person having ordinary skill in the art would be able to convert the sequence of the targets to their RNA form by simply replacing the thymidine (T) with uracil (U) in the sequence.

Table 64 describes a series of oligomeric compounds designed and synthesized to target different regions of pri-miRNAs. These oligomeric compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR, or they can be used in other assays to investigate the role of miRNAs or miRNA downstream targets. In Table 64, “Pri-miRNA” indicates the particular pri-miRNA which contains the miRNA that the oligomeric compound was designed to target. All compounds listed in Table 64 have phosphorothioate internucleoside linkages. In some embodiments, chimeric oligonucleotides (“gapmers”) are composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five nucleotide “wings,” wherein the wings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides. These chimeric compounds are indicated in the “Chemistry” column as “5-10-5 MOE gapmer.” In some embodiments, oligomeric compound consist of 2′-MOE ribonucleotides throughout, and these are indicated by “uniform MOE.”

TABLE 64 Phosphorothioate oligomeric compounds targeting pri-miRNAs SEQ ID ISIS # NO sequence chemistry Pri-miRNA 338615 442 AGAACAGCATGACGTAACCT uniform MOE mir-140, Human 338616 443 GCCCATCTGTGGCTTCACAG uniform MOE mir-30a, Human 338617 444 GAAGTCCGAGGCAGTAGGCA uniform MOE mir-30a, Human 338618 445 CTTCCTTACTATTGCTCACA uniform MOE mir-34, Human 338619 446 GCTAGATACAAAGATGGAAA uniform MOE mir-29b-1, Human 338620 447 CTAGACAATCACTATTTAAA uniform MOE mir-29b-2, Human 338621 448 GCAGCGCAGCTGGTCTCCCC uniform MOE mir-29b-2, Human 338622 449 TAATATATATTTCACTACGC uniform MOE mir-16-3, Human 338623 450 TGCTGTATCCCTGTCACACT uniform MOE mir-16-3, Human 338624 451 CAATTGCGCTACAGAACTGT uniform MOE mir-203, Human 338625 452 TCGATTTAGTTATCTAAAAA uniform MOE mir-7-1, Human 338626 453 CTGTAGAGGCATGGCCTGTG uniform MOE mir-7-1, Human 338627 454 TGACTATACGGATACCACAC uniform MOE mir-10b, Human 338628 455 GGAACAAGGCCAATTATTGC uniform MOE mir-128a, Human 338629 456 AGAAATGTAAACCTCTCAGA uniform MOE mir-128a, Human 338630 457 AGCTGTGAGGGAGAGAGAGA uniform MOE mir-153-1, Human 338631 458 CTGGAGTGAGAATACTAGCT uniform MOE mir-153-1, Human 338632 459 ACTGGGCTCATATTACTAGC uniform MOE mir-153-2, Human 338633 460 TTGGATTAAATAACAACCTA uniform MOE hypothetical miR- 13/miR-190, Human 338634 461 CCCGGAGACAGGGCAAGACA uniform MOE hypothetical miR- 13/miR-190, Human 338635 462 AAAGCGGAAACCAATCACTG uniform MOE chromosome 9 ORF3 containing mir-23b, mir-24-2 and mir-27b, Human 338636 463 GTCCCCATCTCACCTTCTCT uniform MOE chromosome 9 ORF3 containing mir-23b, mir-24-2 and mir-27b, Human 338637 464 TCAGAGCGGAGAGACACAAG uniform MOE mir-96, Human 338638 465 TAGATGCACATATCACTACC uniform MOE miR-17/mir-91, Human 338639 466 CTTGGCTTCCCGAGGCAGCT uniform MOE miR-17/mir-91, Human 338640 467 AGTTTGAAGTGTCACAGCGC uniform MOE mir-123/mir-126, Human 338641 468 GCGTTTTCGATGCGGTGCCG uniform MOE mir-123/mir-126, Human 338642 469 GAGACGCGGGGGCGGGGCGC uniform MOE mir-132, Human 338643 470 TACCTCCAGTTCCCACAGTA uniform MOE mir-132, Human 338644 471 TGTGTTTTCTGACTCAGTCA uniform MOE mir-108-1, Human 338645 472 AGAGCACCTGAGAGCAGCGC uniform MOE chromosome 9 ORF3 containing mir-23b, mir-24-2 and mir-27b, Human 338646 473 TCTTAAGTCACAAATCAGCA uniform MOE chromosome 9 ORF3 containing mir-23b, mir-24-2 and mir-27b, Human 338647 474 TCTCCACAGCGGGCAATGTC uniform MOE let-7i, Human 338648 475 GGCGCGCTGTCCGGGCGGGG uniform MOE mir-212, Human 338649 476 ACTGAGGGCGGCCCGGGCAG uniform MOE mir-212, Human 338650 477 GTCCTCTTGCCCAAGCAACA uniform MOE hypothetical miRNA-023, Human 338651 478 GAAGACCAATACACTCATAC uniform MOE mir-131-2/miR-9, Human 338652 479 CCGAGGGGCAACATCACTGC uniform MOE let-7b, Human 338653 480 TCCATAGCTTAGCAGGTCCA uniform MOE mir-1d-1, Human 338654 481 TTTGATAGTTTAGACACAAA uniform MOE mir-122a, Human 338655 482 GGGAAGGATTGCCTAGCAGT uniform MOE mir-122a, Human 338656 483 AGCTTTAGCTGGGTCAGGAC uniform MOE mir-22, Human 338657 484 TACCATACAGAAACACAGCA uniform MOE mir-92-1, Human 338658 485 TCACAATCCCCACCAAACTC uniform MOE mir-92-1, Human 338659 486 TCACTCCTAAAGGTTCAAGT uniform MOE hypothetical miRNA-30, Human 338660 487 CACCCTCCAGTGCTGTTAGT uniform MOE mir-142, Human 338661 488 CTGACTGAGACTGTTCACAG uniform MOE mir-183, Human 338662 489 CCTTTAGGGGTTGCCACACC uniform MOE glutamate receptor, ionotrophic, AMPA 3/ hypothetical miRNA-033, Human 338663 490 ACAGGTGAGCGGATGTTCTG uniform MOE mir-214, Human 338665 492 AGAGGGGAGACGAGAGCACT uniform MOE mir-192-1, Human 338666 493 TCACGTGGAGAGGAGTTAAA uniform MOE hypothetical miRNA-039, Human 338667 494 AGTGCTAATACTTCTTTCAT uniform MOE hypothetical miRNA-040, Human 338668 495 ACCTGTGTAACAGCCGTGTA uniform MOE hypothetical miRNA-041, Human 338669 496 TTATCGGAACTTCACAGAGA uniform MOE hypothetical miRNA-041, Human 338670 497 TCCCATAGCAGGGCAGAGCC uniform MOE let-7a-3, Human 338671 498 GGCACTTCATTGCTGCTGCC uniform MOE hypothetical miRNA-043, Human 338672 499 GGAGCCTTGCGCTCAGCATT uniform MOE hypothetical miRNA-043, Human 338673 500 ATGGTAATTTCATTTCAGGC uniform MOE hypothetical miRNA-044, Human 338674 501 GATTGCACATCCACACTGTC uniform MOE hypothetical miRNA-044, Human 338675 502 GCTGGCCTGATAGCCCTTCT uniform MOE mir-181a, Human 338676 503 GTTTTTTCAAATCCCAAACT uniform MOE mir-181a, Human 338677 504 CCCAGTGGTGGGTGTGACCC uniform MOE let-7a-1, Human 338678 505 CTGGTTGGGTATGAGACAGA uniform MOE mir-205, Human 338679 506 TTGATCCATATGCAACAAGG uniform MOE mir-103-1, Human 338680 507 GCCATTGGGACCTGCACAGC uniform MOE miR-26a-1, Human 338681 508 ATGGGTACCACCAGAACATG uniform MOE mir-33a, Human 338682 509 AGTTCAAAACTCAATCCCAA uniform MOE mir-196-2, Human 338683 510 GCCCTCGACGAAAACCGACT uniform MOE mir-196-2, Human 338684 511 TTGAACTCCATGCCACAAGG uniform MOE mir-107, Human 338685 512 AGGCCTATTCCTGTAGCAAA uniform MOE mir-106, Human 338686 513 GTAGATCTCAAAAAGCTACC uniform MOE mir-106, Human 338687 514 CTGAACAGGGTAAAATCACT uniform MOE let-7f-1, Human 338688 515 AGCAAGTCTACTCCTCAGGG uniform MOE let-7f-1, Human 338689 516 AATGGAGCCAAGGTGCTGCC uniform MOE hypothetical miRNA-055, Human 338690 517 TAGACAAAAACAGACTCTGA uniform MOE mir-29c, Human 338691 518 GCTAGTGACAGGTGCAGACA uniform MOE mir-130a, Human 338692 519 GGGCCTATCCAAAGTGACAG uniform MOE hypothetical miRNA-058, Human 338693 520 TACCTCTGCAGTATTCTACA uniform MOE hypothetical miRNA-058, Human 338694 521 TTTACTCATACCTCGCAACC uniform MOE mir-218-1, Human 338695 522 AATTGTATGACATTAAATCA uniform MOE mir-124a-2, Human 338696 523 CTTCAAGTGCAGCCGTAGGC uniform MOE mir-124a-2, Human 338697 524 TGCCATGAGATTCAACAGTC uniform MOE mir-21, Human 338698 525 ACATTGCTATCATAAGAGCT uniform MOE mir-16-1, Human 338699 526 TAATTTTAGAATCTTAACGC uniform MOE mir-16-1, Human 338700 527 AGTGTCTCATCGCAAACTTA uniform MOE mir-144, Human 338701 528 TGTTGCCTAACGAACACAGA uniform MOE mir-221, Human 338702 529 GCTGATTACGAAAGACAGGA uniform MOE mir-222, Human 338703 530 GCTTAGCTGTGTCTTACAGC uniform MOE mir-30d, Human 338704 531 GAGGATGTCTGTGAATAGCC uniform MOE mir-30d, Human 338705 532 CCACATATACATATATACGC uniform MOE mir-19b-2, Human 338706 533 AGGAAGCACACATTATCACA uniform MOE mir-19b-2, Human 338707 534 GACCTGCTACTCACTCTCGT uniform MOE mir-128b, Human 338708 535 GGTTGGCCGCAGACTCGTAC uniform MOE hypothetical miRNA 069/mir-219-2, Human 338709 536 GATGTCACTGAGGAAATCAC uniform MOE hypothetical miRNA-070, Human 338710 537 TCAGTTGGAGGCAAAAACCC uniform MOE LOC 114614/ hypothetical miRNA-071, Human 338711 538 GGTAGTGCAGCGCAGCTGGT uniform MOE mir-29b-2, Human 338712 539 CCGGCTATTGAGTTATGTAC uniform MOE mir-129-2, Human 338713 540 ACCTCTCAGGAAGACGGACT uniform MOE mir-133b, Human 338714 541 GAGCATGCAACACTCTGTGC uniform MOE hypothetical miRNA-075, Human 338715 542 CCTCCTTGTGGGCAAAATCC uniform MOE let-7d, Human 338716 543 CGCATCTTGACTGTAGCATG uniform MOE mir-15b, Human 338717 544 TCTAAGGGGTCACAGAAGGT uniform MOE mir-29a-1, Human 338718 545 GAAAATTATATTGACTCTGA uniform MOE mir-29a-1, Human 338719 546 GGTTCCTAATTAAACAACCC uniform MOE hypothetical miRNA-079, Human 338720 547 CCGAGGGTCTAACCCAGCCC uniform MOE mir-199b, Human 338721 548 GACTACTGTTGAGAGGAACA uniform MOE mir-129-1, Human 338722 549 TCTCCTTGGGTGTCCTCCTC uniform MOE let-7e, Human 338723 550 TGCTGACTGCTCGCCCTTGC uniform MOE hypothetical miRNA-083, Human 338724 551 ACTCCCAGGGTGTAACTCTA uniform MOE let7c-1, Human 338725 552 CATGAAGAAAGACTGTAGCC uniform MOE mir-204, Human 338726 553 GACAAGGTGGGAGCGAGTGG uniform MOE mir-145, Human 338727 554 TGCTCAGCCAGCCCCATTCT uniform MOE mir-124a-1, Human 338728 555 GCTTTTAGAACCACTGCCTC uniform MOE DiGeorge syndrome critical region gene 8/ hypothetical miRNA-088, Human 338729 556 GGAGTAGATGATGGTTAGCC uniform MOE mir-213/mir-181a, Human 338730 557 ACTGATTCAAGAGCTTTGTA uniform MOE hypothetical miRNA-090, Human 338731 558 GTAGATAACTAAACACTACC uniform MOE mir-20, Human 338732 559 AATCCATTGAAGAGGCGATT uniform MOE mir-133a-1, Human 338733 560 GGTAAGAGGATGCGCTGCTC uniform MOE mir-138-2, Human 338734 561 GGCCTAATATCCCTACCCCA uniform MOE mir-98, Human 338735 562 GTGTTCAGAAACCCAGGCCC uniform MOE mir-196-1, Human 338736 563 TCCAGGATGCAAAAGCACGA uniform MOE mir-125b-1, Human 338737 564 TACAACGGCATTGTCCTGAA uniform MOE mir-199a-2, Human 338738 565 TTTCAGGCTCACCTCCCCAG uniform MOE hypothetical miRNA-099, Human 338739 566 AAAAATAATCTCTGCACAGG uniform MOE mir-181b, Human 338740 567 AGAATGAGTTGACATACCAA uniform MOE hypothetical miRNA-101, Human 338741 568 GCTTCACAATTAGACCATCC uniform MOE mir-141, Human 338742 569 AGACTCCACACCACTCATAC uniform MOE mir-131-1/miR-9, Human 338743 570 ATCCATTGGACAGTCGATTT uniform MOE mir-133a-2, Human 338744 571 GGCGGGCGGCTCTGAGGCGG uniform MOE hypothetical miRNA-105, Human 338745 572 CTCTTTAGGCCAGATCCTCA uniform MOE hypothetical miRNA-105, Human 338746 573 TAATGGTATGTGTGGTGATA uniform MOE hypothetical miRNA-107, Human 338747 574 ATTACTAAGTTGTTAGCTGT uniform MOE miR-1d-2, Human 338748 575 GATGCTAATCTACTTCACTA uniform MOE mir-18, Human 338749 576 TCAGCATGGTGCCCTCGCCC uniform MOE mir-220, Human 338750 577 TCCGCGGGGGCGGGGAGGCT uniform MOE hypothetical miRNA-111, Human 338751 578 AGACCACAGCCACTCTAATC uniform MOE mir-7-3, Human 338752 579 TCCGTTTCCATCGTTCCACC uniform MOE mir-218-2, Human 338753 580 GCCAGTGTACACAAACCAAC uniform MOE mir-24-2, Human 338754 581 AAGGCTTTTTGCTCAAGGGC uniform MOE chromosome 9 ORF3 containing mir-23b, mir-24-2 and mir-27b, Human 338755 582 TTGACCTGAATGCTACAAGG uniform MOE mir-103-2, Human 338756 583 TGCCCTGCTCAGAGCCCTAG uniform MOE mir-211, Human 338757 584 TCAATGTGATGGCACCACCA uniform MOE mir-101-3, Human 338758 585 ACCTCCCAGCCAATCCATGT uniform MOE mir-30b, Human 338759 586 TCCTGGATGATATCTACCTC uniform MOE hypothetical miRNA-120, Human 338760 587 TCTCCCTTGATGTAATTCTA uniform MOE let-7a-4, Human 338761 588 AGAGCGGAGTGTTTATGTCA uniform MOE mir-10a, Human 338762 589 TCATTCATTTGAAGGAAATA uniform MOE mir-19a, Human 338763 590 TCCAAGATGGGGTATGACCC uniform MOE let-7f-2, Human 338764 591 TTTTTAAACACACATTCGCG uniform MOE mir-15a-1, Human 338765 592 AGATGTGTTTCCATTCCACT uniform MOE mir-108-2, Human 338766 593 CCCCCTGCCGCTGGTACTCT uniform MOE mir-137, Human 338767 594 CGGCCGGAGCCATAGACTCG uniform MOE mir-219-1, Human 338768 595 CTTTCAGAGAGCCACAGCCT uniform MOE mir-148b, Human 338769 596 GCTTCCCAGCGGCCTATAGT uniform MOE mir-130b, Human 338770 597 CAGCAGAATATCACACAGCT uniform MOE mir-19b-1, Human 338771 598 TACAATTTGGGAGTCCTGAA uniform MOE mir-199b, Human 338772 599 GCCTCCTTCATATATTCTCA uniform MOE mir-204, Human 338773 600 CCCCATCTTAGCATCTAAGG uniform MOE mir-145, Human 338774 601 TTGTATGGACATTTAAATCA uniform MOE mir-124a-1, Human 338775 602 TTTGATTTTAATTCCAAACT uniform MOE mir-213/mir-181a, Human 338776 603 CAAACGGTAAGATTTGCAGA uniform MOE hypothetical miRNA-090, Human 338777 604 GGATTTAAACGGTAAACATC uniform MOE mir-125b-1, Human 338778 605 CTCTAGCTCCCTCACCAGTG uniform MOE hypothetical miRNA-099, Human 338779 606 GCTTGTCCACACAGTTCAAC uniform MOE mir-181b, Human 338780 607 GCATTGTATGTTCATATGGG uniform MOE miR-1d-2, Human 338781 608 TGTCGTAGTACATCAGAACA uniform MOE mir-7-3, Human 338782 609 AGCCAGTGTGTAAAATGAGA uniform MOE chromosome 9 ORF3 containing mir-23b, mir-24-2 and mir-27b, Human 338783 610 TTCAGATATACAGCATCGGT uniform MOE mir-101-3, Human 338784 611 TGACCACAAAATTCCTTACA uniform MOE mir-10a, Human 338785 612 ACAACTACATTCTTCTTGTA uniform MOE mir-19a, Human 338786 613 TGCACCTTTTCAAAATCCAC uniform MOE mir-15a-1, Human 338787 614 AACGTAATCCGTATTATCCA uniform MOE mir-137, Human 338788 615 CGTGAGGGCTAGGAAATTGC uniform MOE mir-216, Human 338789 616 GCAACAGGCCTCAATATCTT uniform MOE mir-100-1, Human 338790 617 ACGAGGGGTCAGAGCAGCGC uniform MOE mir-187, Human 338791 618 GGCAGACGAAAGGCTGACAG uniform MOE hypothetical miRNA-137, Human 338792 619 CTGCACCATGTTCGGCTCCC uniform MOE hypothetical miRNA-138, Human 338793 620 GGGGCCCTCAGGGCTGGGGC uniform MOE mir-124a-3, Human 338794 621 CCGGTCCACTCTGTATCCAG uniform MOE mir-7-2, Human 338795 622 GCTGGGAAAGAGAGGGCAGA uniform MOE hypothetical miRNA-142, Human 338796 623 TCAGATTGCCAACATTGTGA uniform MOE hypothetical miRNA-143, Human 338797 624 CTGGGGAGGGGGTTAGCGTC uniform MOE collagen, type I, alpha 1/hypothetical miRNA- 144, Human 338798 625 TGGGTCTGGGGCAGCGCAGT uniform MOE mir-210, Human 338799 626 TTGAAGTAGCACAGTCATAC uniform MOE mir-215, Human 338800 627 TCTACCACATGGAGTGTCCA uniform MOE mir-223, Human 338801 628 AGTGCCGCTGCCGCGCCGTG uniform MOE mir-131-3/miR-9, Human 338802 629 ACACATTGAGAGCCTCCTGA uniform MOE mir-199a-1, Human 338803 630 GTCGCTCAGTGCTCTCTAGG uniform MOE mir-30c-1, Human 338804 631 AGGCTCCTCTGATGGAAGGT uniform MOE mir-101-1, Human 338805 632 GCTGTGACTTCTGATATTAT uniform MOE hypothetical miRNA-153, Human 338806 633 GACATCATGTGATTTGCTCA uniform MOE hypothetical miRNA-154, Human 338807 634 CACCCCAAGGCTGCAGGGCA uniform MOE mir-26b, Human 338808 635 TGTCAAGCCTGGTACCACCA uniform MOE hypothetical miRNA-156, Human 338809 636 CTGCTCCAGAGCCCGAGTCG uniform MOE mir-152, Human 338810 637 ACCCTCCGCTGGCTGTCCCC uniform MOE mir-135-1, Human 338811 638 TAGAGTGAATTTATCTTGGT uniform MOE non-coding RNA in rhabdomyosarcoma/mir- 135-2, Human 338812 639 TGGTGACTGATTCTTATCCA uniform MOE mir-217, Human 338813 640 CAATATGATTGGATAGAGGA uniform MOE hypothetical miRNA-161, Human 338814 641 TTTAAACACACATTCGCGCC uniform MOE mir-15a-1, Human 338815 642 ACCGGGTGGTATCATAGACC uniform MOE let-7g, Human 338816 643 TGCATACCTGTTCAGTTGGA uniform MOE hypothetical miRNA-164, Human 338817 644 GCCCGCCTCTCTCGGCCCCC uniform MOE sterol regulatory element-binding protein-1/mir-33b, Human 338818 645 TCGCCCCCTCCCAGGCCTCT uniform MOE hypothetical miRNA-166, Human 338819 646 ACAACTGTAGAGTATGGTCA uniform MOE mir-16-1, Human 338820 647 GCTGACCATCAGTACTTTCC uniform MOE hypothetical miRNA 168- 1/similar to ribosomal protein L5, Human 338821 648 TTATAGAACAGCCTCCAGTG uniform MOE forkhead box P2/hypothetical miRNA- 169, Human 338822 649 TTCAGGCACTAGCAGTGGGT uniform MOE hypothetical miRNA-170, Human 338823 650 AGTACTGCGAGGTTAACCGC uniform MOE glutamate receptor, ionotropic, AMPA 2/ hypothetical miRNA-171, Human 338824 651 GGACCTTTAAGATGCAAAGT uniform MOE hypothetical miRNA-172, Human 338825 652 TTCATATTATCCACCCAGGT uniform MOE hypothetical miRNA-173, Human 338826 653 CGGATCCTGTTACCTCACCA uniform MOE mir-182, Human 338827 654 TGGTGCCTGCCACATCTTTG uniform MOE hypothetical miRNA-175, Human 338828 655 TGGGAGGCTGAATCAAGGAC uniform MOE hypothetical miRNA-176, Human 338829 656 TGACAACCAGGAAGCTTGTG uniform MOE hypothetical miRNA-177- 1, Human 338830 657 GCCAGGCAGCGAGCTTTTGA uniform MOE hypothetical miRNA-178, Human 338831 658 CAGCCTGCCACCGCCGCTTT uniform MOE hypothetical miRNA-179, Human 338832 659 CTGCCCCCGTGGACCGAACA uniform MOE cezanne 2/hypothetical miRNA-180, Human 338833 660 TCGTGCACCTGAGGAGTCTG uniform MOE hypothetical miRNA-181, Human 338834 661 CAAACGTGCTGTCTTCCTCC uniform MOE mir-148a, Human 338835 662 AAGGACTCAGCAGTGTTTCA uniform MOE tight junction protein 1 (zona occludens 1)/ hypothetical miRNA-183, Human 338836 663 TCCTCGGTGGCAGAGCTCAG uniform MOE mir-23a, Human 338837 664 AGACAATGAGTACACAGTTC uniform MOE hypothetical miRNA-185, Human 338838 665 CTGCAAGCACTGGTTCCCAT uniform MOE hypothetical miRNA-177- 2/hypothetical miRNA 186, Human 338839 666 TTGCCTGAGCTGCCCAAACT uniform MOE mir-181c, Human 338840 667 TCCATCACACTGTCCTATGA uniform MOE hypothetical miRNA-188, Human 338841 668 GAGGGATTGTATGAACATCT uniform MOE mir-216, Human 338842 669 GCTTGTGCGGACTAATACCA uniform MOE mir-100-1, Human 338843 670 GCAGGCTAAAAGAAATAAGC uniform MOE hypothetical miRNA-138, Human 338844 671 ATTGTATAGACATTAAATCA uniform MOE mir-124a-3, Human 338845 672 GTTGAGCGCAGTAAGACAAC uniform MOE mir-7-2, Human 338846 673 AGATGTTTCTGGCCTGCGAG uniform MOE hypothetical miRNA-142, Human 338847 674 GACAAACTCAGCTATATTGT uniform MOE mir-215, Human 338848 675 ACGGCTCTGTGGCACTCATA uniform MOE mir-131-3/miR-9, Human 338849 676 GCTTTCTTACTTTCCACAGC uniform MOE mir-30c-1, Human 338850 677 TACCTTTAGAATAGACAGCA uniform MOE mir-101-1, Human 338851 678 AGGCTGGACAGCACACAACC uniform MOE mir-26b, Human 338852 679 AGCAGGAGCCTTATCTCTCC uniform MOE hypothetical miRNA-156, Human 338853 680 ATGAGTGAGCAGTAGAATCA uniform MOE mir-135-1, Human 338854 681 TGAGACTTTATTACTATCAC uniform MOE non-coding RNA in rhabdomyosarcoma/mir- 135-2, Human 338855 682 TACTTTACTCCAAGGTTTTA uniform MOE mir-15a-1, Human 338856 683 GCACCCGCCTCACACACGTG uniform MOE sterol regulatory element-binding protein-1/mir-33b, Human 338857 684 TTCCCGACCTGCCTTTACCT uniform MOE hypothetical miRNA-166, Human 338858 685 TCCTGTAATTATAGGCTAGC uniform MOE forkhead box P2/hypothetical miRNA- 169, Human 338859 686 GGATCATATCAATAATACCA uniform MOE hypothetical miRNA-172, Human 338860 687 TGCTGAGACACACAATATGT uniform MOE hypothetical miRNA-176, Human 338861 688 TGTTTGTCTCCAAGAAACGT uniform MOE hypothetical miRNA-177- 1, Human 338862 689 TGTCATGGACAGGATGAATA uniform MOE hypothetical miRNA-179, Human 338863 690 TCTATCATACTCAGAGTCGG uniform MOE mir-148a, Human 338864 691 TTGTGACAGGAAGCAAATCC uniform MOE mir-23a, Human 338865 692 CATCAGAGTCACCAACCCCA uniform MOE hypothetical miRNA-185, Human 338866 693 CAAGAGATGTCTCGTTTTGC uniform MOE hypothetical miRNA-177- 2/hypothetical miRNA 186, Human 340342 937 GACTGTTGAATCTCATGGCA uniform MOE miR-104 (Mourelatos), Human 340344 1656 GCATGAGCAGCCACCACAGG uniform MOE miR-105 (Mourelatos), Human 340346 1626 ACGACTTGGTGTGGACCCTG uniform MOE miR-27 (Mourelatos), Human 340347 849 TACTTTATATAGAACACAAG uniform MOE mir-92-2/miR-92 (Mourelatos), Human 340349 1632 AGGTTGGGTAATCACACTAC uniform MOE miR-93 (Mourelatos), Human 340351 1621 AATGTAACGCATTTCAATTC uniform MOE miR-95 (Mourelatos), Human 340353 1694 TGTGCGGTCCACTTCACCAC uniform MOE miR-99 (Mourelatos), Human 340355 1671 GTCCAGCAATTGCCCAAGTC uniform MOE miR-25, Human 340357 1662 GGAAAGTCAGAAAGGTAACT uniform MOE miR-28, Human 340359 1635 CAGGTTCCCAGTTCAACAGC uniform MOE miR-31, Human 340361 1636 CATTGAGGCCGTGACAACAT uniform MOE miR-32, Human 340363 1656 GCATGAGCAGCCACCACAGG 5-10-5 MOE miR-105 (Mourelatos), gapmer Human 340364 1626 ACGACTTGGTGTGGACCCTG 5-10-5 MOE miR-27 (Mourelatos), gapmer Human 340366 1632 AGGTTGGGTAATCACACTAC 5-10-5 MOE miR-93 (Mourelatos), gapmer Human 340367 1621 AATGTAACGCATTTCAATTC 5-10-5 MOE miR-95 (Mourelatos), gapmer Human 340368 1694 TGTGCGGTCCACTTCACCAC 5-10-5 MOE miR-99 (Mourelatos), gapmer Human 340369 1671 GTCCAGCAATTGCCCAAGTC 5-10-5 MOE miR-25, Human gapmer 340370 1662 GGAAAGTCAGAAAGGTAACT 5-10-5 MOE miR-28, Human gapmer 340371 1635 CAGGTTCCCAGTTCAACAGC 5-10-5 MOE miR-31, Human gapmer 340372 1636 CATTGAGGCCGTGACAACAT 5-10-5 MOE miR-32, Human gapmer 341817 1630 AGCCACCTTGAGCTCACAGC uniform MOE miR-30c-2, Human 341818 1695 TGTGTGCGGCGAAGGCCCCG uniform MOE miR-99b, Human 341819 1657 GCCAGGCTCCCAAGAACCTC uniform MOE MiR-125a, Human 341820 1653 GATGTTACTAAAATACCTCA uniform MOE MiR-125b-2, Human 341822 1679 TCCGATGATCTTTCTGAATC uniform MOE miR-127, Human 341825 1646 CTTAAAATAAAACCAGAAAG uniform MOE miR-186, Human 341826 1618 AAAATCACAGGAACCTATCT uniform MOE miR-198, Human 341827 1688 TGGAATGCTCTGGAGACAAC uniform MOE miR-191, Human 341828 1677 TCCATAGCAAAGTAATCCAT uniform MOE miR-206, Human 341829 1668 GGTAGCACGGAGAGGACCAC uniform MOE miR-94, Human 341830 1624 ACACTTACAGTCACAAAGCT uniform MOE miR-184, Human 341831 1654 GCAGACTCGCTTCCCTGTGC uniform MOE miR-195, Human 341832 1684 TGATCCGACACCCTCATCTC uniform MOE miR-193, Human 341833 1641 CCTGGGGAGGGGACCATCAG uniform MOE miR-185, Human 341834 1676 TCAGAAAGCTCACCCTCCAC uniform MOE miR-188, Human 341835 1648 GAGCTCTTACCTCCCACTGC uniform MOE miR-197, Human 341836 1686 TGGAAATTGGTACACAGTCC uniform MOE miR-194-1, Human 341837 1642 CGTGAGCATCAGGTATAACC uniform MOE miR-208, Human 341838 1687 TGGAACCAGTGGGCACTTCC uniform MOE miR-194-2, Human 341839 1638 CCAGCCTCCGAGCCACACTG uniform MOE miR-139, Human 341840 1628 AGACCTGACTCCATCCAATG uniform MOE miR-200b, Human 341841 1629 AGAGTCAAGCTGGGAAATCC uniform MOE miR-200a, Human 341843 1630 AGCCACCTTGAGCTCACAGC 5-10-5 MOE miR-30c-2, Human gapmer 341844 1695 TGTGTGCGGCGAAGGCCCCG 5-10-5 MOE miR-99b, Human gapmer 341845 1657 GCCAGGCTCCCAAGAACCTC 5-10-5 MOE MiR-125a, Human gapmer 341846 1653 GATGTTACTAAAATACCTCA 5-10-5 MOE MiR-125b-2, Human gapmer 341848 1679 TCCGATGATCTTTCTGAATC 5-10-5 MOE miR-127, Human gapmer 341851 1646 CTTAAAATAAAACCAGAAAG 5-10-5 MOE miR-186, Human gapmer 341852 1618 AAAATCACAGGAACCTATCT 5-10-5 MOE miR-198, Human gapmer 341853 1688 TGGAATGCTCTGGAGACAAC 5-10-5 MOE miR-191, Human gapmer 341854 1677 TCCATAGCAAAGTAATCCAT 5-10-5 MOE miR-206, Human gapmer 341855 1668 GGTAGCACGGAGAGGACCAC 5-10-5 MOE miR-94, Human gapmer 341856 1624 ACACTTACAGTCACAAAGCT 5-10-5 MOE miR-184, Human gapmer 341857 1654 GCAGACTCGCTTCCCTGTGC 5-10-5 MOE miR-195, Human gapmer 341858 1684 TGATCCGACACCCTCATCTC 5-10-5 MOE miR-193, Human gapmer 341859 1641 CCTGGGGAGGGGACCATCAG 5-10-5 MOE miR-185, Human gapmer 341860 1676 TCAGAAAGCTCACCCTCCAC 5-10-5 MOE miR-188, Human gapmer 341861 1648 GAGCTCTTACCTCCCACTGC 5-10-5 MOE miR-197, Human gapmer 341862 1686 TGGAAATTGGTACACAGTCC 5-10-5 MOE miR-194-1, Human gapmer 341863 1642 CGTGAGCATCAGGTATAACC 5-10-5 MOE miR-208, Human gapmer 341864 1687 TGGAACCAGTGGGCACTTCC 5-10-5 MOE miR-194-2, Human gapmer 341865 1638 CCAGCCTCCGAGCCACACTG 5-10-5 MOE miR-139, Human gapmer 341866 1628 AGACCTGACTCCATCCAATG 5-10-5 MOE miR-200b, Human gapmer 341867 1629 AGAGTCAAGCTGGGAAATCC 5-10-5 MOE miR-200a, Human gapmer 344731 1619 AACGGTTTATGACAAACATT uniform MOE mir-240* (Kosik), Human 344732 1665 GGGCTGTATGCACTTTCTCC uniform MOE mir-232* (Kosik), Human 344733 1667 GGGTCTCCAGCTTTACACCA uniform MOE mir-227* (Kosik)/mir- 226* (Kosik), Human 344734 1649 GAGTCGCCTGAGTCATCACT uniform MOE mir-244* (Kosik), Human 344735 1658 GCCATAAATAAAGCGAACGC uniform MOE mir-224* (Kosik), Human 344736 1678 TCCATTAACCATGTCCCTCA uniform MOE mir-248* (Kosik), Human 344737 1619 AACGGTTTATGACAAACATT 5-10-5 MOE mir-240* (Kosik), Human gapmer 344738 1665 GGGCTGTATGCACTTTCTCC 5-10-5 MOE mir-232* (Kosik), Human gapmer 344739 1667 GGGTCTCCAGCTTTACACCA 5-10-5 MOE mir-227* (Kosik)/mir- gapmer 226* (Kosik), Human 344740 1649 GAGTCGCCTGAGTCATCACT 5-10-5 MOE mir-244* (Kosik), Human gapmer 344741 1658 GCCATAAATAAAGCGAACGC 5-10-5 MOE mir-224* (Kosik), Human gapmer 344742 1678 TCCATTAACCATGTCCCTCA 5-10-5 MOE mir-248* (Kosik), Human gapmer 346787 1689 TGGCTTCCATAGTCTGGTGT uniform MOE miR-147 (Sanger), Human 346788 1623 ACAATGCACAATCATCTACT uniform MOE miR-224 (Sanger), Human 346789 1669 GGTGAACACAGTGCATGCCC uniform MOE miR-134 (Sanger), Human 346790 1682 TCTGACACTGACACAACCCA uniform MOE miR-146 (Sanger), Human 346791 1631 AGGGTCTGAGCCCAGCACTG uniform MOE miR-150 (Sanger), Human 346792 1637 CCAAGAGACGTTTCATTTTG uniform MOE hypothetical miRNA-177- 3, Human 346793 1683 TCTGATTGGCAACGGCCTGA uniform MOE mir-138-3, Human 346794 1627 ACTGTCCATCTTAGTTCAGA uniform MOE mir-138-4, Human 346795 1634 AGTTGATTCAGACTCAAACC uniform MOE mir-181b-2, Human 346796 1655 GCATAAGCAGCCACCACAGG uniform MOE miR-105-2, Human 346797 1691 TGTATGATATCTACCTCAGG uniform MOE hypothetical miRNA-120- 2, Human 346798 1689 TGGCTTCCATAGTCTGGTGT 5-10-5 MOE miR-147 (Sanger), Human gapmer 346799 1623 ACAATGCACAATCATCTACT 5-10-5 MOE miR-224 (Sanger), Human gapmer 346800 1669 GGTGAACACAGTGCATGCCC 5-10-5 MOE miR-134 (Sanger), Human gapmer 346801 1682 TCTGACACTGACACAACCCA 5-10-5 MOE miR-146 (Sanger), Human gapmer 346802 1631 AGGGTCTGAGCCCAGCACTG 5-10-5 MOE miR-150 (Sanger), Human gapmer 346803 1637 CCAAGAGACGTTTCATTTTG 5-10-5 MOE hypothetical miRNA-177- gapmer 3, Human 346804 1683 TCTGATTGGCAACGGCCTGA 5-10-5 MOE mir-138-3, Human gapmer 346805 1627 ACTGTCCATCTTAGTTCAGA 5-10-5 MOE mir-138-4, Human gapmer 346806 1634 AGTTGATTCAGACTCAAACC 5-10-5 MOE mir-181b-2, Human gapmer 346807 1655 GCATAAGCAGCCACCACAGG 5-10-5 MOE miR-105-2, Human gapmer 346808 1691 TGTATGATATCTACCTCAGG 5-10-5 MOE hypothetical miRNA-120- gapmer 2, Human 348225 1620 AAGAGAAGGCGGAGGGGAGC 5-10-5 MOE miR-320, Human gapmer 348226 1643 CTCGAACCCACAATCCCTGG 5-10-5 MOE miR-321-1, Human gapmer 354006 1650 GAGTTTGGGACAGCAATCAC 5-10-5 MOE mir-135b (Ruvkun), gapmer Human 354007 1633 AGTAGGGGATGAGACATACT 5-10-5 MOE mir-151* (Ruvkun), gapmer Human 354008 1639 CCCACAAACGACATATGACA 5-10-5 MOE mir-340 (Ruvkun), Human gapmer 354009 1664 GGCCTGGTTTGATCTGGGAT 5-10-5 MOE mir-331 (Ruvkun), Human gapmer 354010 1647 GAGACTCCCAACCGCACCCA 5-10-5 MOE miR-200c (RFAM-Human) gapmer 354011 1700 TTGTAACCACCACAGTACAA 5-10-5 MOE miR-34b (RFAM-Human) gapmer 354012 1663 GGAGGACAGGGAGAGCGGCC 5-10-5 MOE mir-339-1 (RFAM-Human) gapmer 354013 1675 TCACAGGCAGGCACACGTGA 5-10-5 MOE mir-339-1 (RFAM-Human) gapmer 354014 1698 TTCAGAGCTACAGCATCGGT 5-10-5 MOE mir-101-3, Mouse gapmer 354015 1670 GTAGAACTCAAAAAGCTACC 5-10-5 MOE mir-106, Mouse gapmer 354016 1673 TAGATGCACACATCACTACC 5-10-5 MOE miR-17/mir-91, Mouse gapmer 354017 1690 TGTACAATTTGGGAGTCCTG 5-10-5 MOE mir-199b, Human gapmer 354018 1644 CTCTTTAGACCAGATCCACA 5-10-5 MOE hypothetical miRNA-105, gapmer Mouse 354019 1640 CCTCACTCAGAGGCCTAGGC 5-10-5 MOE mir-211, Mouse gapmer 354020 1666 GGGGATTAAGTCTTATCCAG 5-10-5 MOE mir-217, Mouse gapmer 354021 1622 ACAATGCACAAACCATCTAC 5-10-5 MOE miR-224 (Sanger), Mouse gapmer 354022 1693 TGTCATATCATATCAGAACA 5-10-5 MOE mir-7-3, Mouse gapmer 354023 1672 TAGATGACGACACACTACCT 5-10-5 MOE mir-20, Rat gapmer 354024 1692 TGTCACAAACACTTACTGGA 5-10-5 MOE mir-325 (Ruvkun), Human gapmer 354025 1625 ACGAATTATGTCACAAACAC 5-10-5 MOE mir-325 (Ruvkun), Mouse gapmer 354026 1651 GATCTGAGCACCACCCGCCT 5-10-5 MOE mir-326 (Ruvkun), Human gapmer 354027 1652 GATCTGAGCATAACCCGCCT 5-10-5 MOE mir-326 (Ruvkun), Mouse gapmer 354028 1697 TGTTTCGTCCTCATTAAAGA 5-10-5 MOE mir-329-1 (Ruvkun), gapmer Human 354029 1699 TTCTCATCAAAGAAACAGAG 5-10-5 MOE mir-329-1 (Ruvkun), gapmer Mouse 354030 1696 TGTTTCGTCCTCAATAAAGA 5-10-5 MOE mir-329-2 (Ruvkun), gapmer Human 354031 1681 TCGGTTGATCTTGCAGAGCC 5-10-5 MOE mir-330 (Ruvkun), Human gapmer 354032 1685 TGCTCGTTGGATCTTGAAGA 5-10-5 MOE mir-330 (Ruvkun), Mouse gapmer 354033 1661 GCTGGATAACTGTGCATCAA 5-10-5 MOE mir-337 (Ruvkun), Human gapmer 354034 1645 CTGAATGGCTGTGCAATCAA 5-10-5 MOE mir-337 (Ruvkun), Mouse gapmer 354035 1659 GCCCACCAGCCATCACGAGC 5-10-5 MOE mir-345 (Ruvkun), Human gapmer 354036 1660 GCCCAGTAGCCACCACAAGC 5-10-5 MOE mir-345 (Ruvkun), Mouse gapmer 354037 1680 TCCTTCAGAGCAACAGAGAG 5-10-5 MOE mir-346 (Ruvkun), Human gapmer 354038 1674 TAGTAGGGAGGAGACATACT 5-10-5 MOE mir-151* (Ruvkun), gapmer Mouse 354039 1701 TTGTCAGCACCGCACTACAA 5-10-5 MOE miR-34b (RFAM-Mouse) gapmer

In accordance with the present invention, a further series of oligomeric compounds were designed and synthesized to target different regions of miRNAs. These oligomeric compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR, or they can be used in other assays to investigate the role of miRNAs or miRNA downstream targets. The compounds are shown in Table 65, where “pri-miRNA” indicates the particular pri-miRNA which contains the miRNA that the oligomeric compound was designed to target. Oligomeric compounds having phosphorothioate internucleoside linkages are indicated by “PS” in the “Chemistry” column of Table 65, whereas compounds having phosphodiester internucleoside linkages are indicated by “PO.” In some embodiments, chimeric oligonucleotides (“gapmers”) are composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by nucleotide “wings” two to ten nucleotides in length. The wings are composed of 2′-methoxyethoxy (2′-MOE) ribonucleotides. In some embodiments, chimeric oligonucleotides are of the “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Chimeric oligonucleotides of this type are also known in the art and are indicated in Table 65 as “hemimers.” For example, “PO/6MOE-10deoxy hemimer,” describes a chimeric oligomeric compound consisting of six 2′-MOE ribonucleotides at the 5′-terminus, followed by ten deoxyribonucleotides on the 3′-terminal end, with a phosphodiester backbone throughout the hemimer.

TABLE 65 Oligomeric compounds targeting miRNAs SEQ ID ISIS # NO sequence Chemistry Pri-miRNA 340343 1780 ACAGGAGTCTGAGCATTTGA PS/MOE miR-105 (Mourelatos) 340345 1882 GGAACTTAGCCACTGTGAA PS/MOE miR-27 (Mourelatos) 340350 855 TGCTCAATAAATACCCGTTGAA PS/MOE miR-95 (Mourelatos) 340352 1821 CACAAGATCGGATCTACGGGTT PS/MOE miR-99 (Mourelatos) 340354 1903 TCAGACCGAGACAAGTGCAATG PS/MOE miR-25 (Tuschl) 340356 1853 CTCAATAGACTGTGAGCTCCTT PS/MOE miR-28 (Tuschl) 340358 1825 CAGCTATGCCAGCATCTTGCC PS/MOE miR-31 (Tuschl) 340360 1865 GCAACTTAGTAATGTGCAATA PS/MOE miR-32 (Tuschl) 340924 298 ACAAATTCGGTTCTACAGGGTA PS/MOE 5-10-7 mir-10b gapmer 340925 307 GTGGTAATCCCTGGCAATGTGAT PS/MOE 5-10-8 mir-23b gapmer 340928 322 ACTCACCGACAGCGTTGAATGTT PS/MOE 5-10-8 mir-181a gapmer 340929 331 AACCGATTTCAAATGGTGCTAG PS/MOE 5-10-7 mir-29c gapmer 340930 342 GCAAGCCCAGACCGCAAAAAG PS/MOE 5-10-6 mir-129 gapmer 340931 346 AACCGATTTCAGATGGTGCTAG PS/MOE 5-10-7 mir-29a gapmer 340932 349 AACCATACAACCTACTACCTCA PS/MOE 5-10-7 let-7c gapmer 340933 352 GGTACAATCAACGGTCGATGGT PS/MOE 5-10-7 mir-213 gapmer 340934 356 AACAATACAACTTACTACCTCA PS/MOE 5-10-7 mir-98 gapmer 340935 373 GCCCTTTCATCATTGCACTG PS/MOE 5-10-5 mir-130b gapmer 340936 385 ACTGTACAAACTACTACCTCA PS/MOE 5-10-6 let-7g gapmer 341785 854 GGAGTGAAGACACGGAGCCAGA PS/MOE miR-149 341786 1845 CGCAAGGTCGGTTCTACGGGTG PS/MOE miR-99b 341787 852 CACAGGTTAAAGGGTCTCAGGGA PS/MOE miR-125a 341788 853 AGCCAAGCTCAGACGGATCCGA PS/MOE miR-127 341789 1909 TCCATCATCAAAACAAATGGAGT PS/MOE miR-136 341790 1843 CGAAGGCAACACGGATAACCTA PS/MOE miR-154 341791 1880 GCTTCCAGTCGAGGATGTTTACA PS/MOE miR-30a-s 341792 1911 TCCGTGGTTCTACCCTGTGGTA PS/MOE miR-140-as 341793 1836 CCATAAAGTAGGAAACACTACA PS/MOE miR-142-as 341794 1761 AACAGGTAGTCTGAACACTGGG PS/MOE miR-199-s 341795 1762 AACCAATGTGCAGACTACTGTA PS/MOE miR-199-as 341796 1904 TCATACAGCTAGATAACCAAAGA PS/MOE miR-9 341797 1773 ACAAGTGCCTTCACTGCAGT PS/MOE miR-17 341798 1871 GCATTATTACTCACGGTACGA PS/MOE miR-126a 341799 1787 ACCTAATATATCAAACATATCA PS/MOE miR-190 341800 1766 AAGCCCAAAAGGAGAATTCTTTG PS/MOE miR-186 341801 1839 CCTATCTCCCCTCTGGACC PS/MOE miR-198a 341802 1806 AGCTGCTTTTGGGATTCCGTTG PS/MOE miR-191c 341803 760 CCACACACTTCCTTACATTCCA PS/MOE miR-206d 341804 761 ATCTGCACTGTCAGCACTTT PS/MOE miR-94 341805 762 ACCCTTATCAGTTCTCCGTCCA PS/MOE miR-184 341806 763 GCCAATATTTCTGTGCTGCTA PS/MOE miR-195 341807 764 CTGGGACTTTGTAGGCCAGTT PS/MOE miR-193 341808 1861 GAACTGCCTTTCTCTCCA PS/MOE miR-185 341809 1786 ACCCTCCACCATGCAAGGGATG PS/MOE miR-188 341810 1879 GCTGGGTGGAGAAGGTGGTGAA PS/MOE miR-197a 341811 1906 TCCACATGGAGTTGCTGTTACA PS/MOE miR-194 341812 1771 ACAAGCTTTTTGCTCGTCTTAT PS/MOE miR-208 341814 1887 GTCATCATTACCAGGCAGTATTA PS/MOE miR-200b 341815 1831 CATCGTTACCAGACAGTGTTA PS/MOE miR-200a 342946 1897 TAGGAGAGAGAAAAAGACTGA PS/MOE miR-14 342947 1827 CAGCTTTCAAAATGATCTCAC PS/MOE miR-Bantam 343875 321 AACTATACAACCTACTACCTCA PO/MOE let-7a 344267 1769 ACAAATTCGGATCTACAGGGTA PS/MOE miR-10 (Tuschl) 344268 1774 ACACAAATTCGGTTCTACAGGG PS/MOE miR-10b (Tuschl) 344269 1890 TAACCGATTTCAAATGGTGCTA PS/MOE miR-29c (Tuschl) 344270 1867 GCACGAACAGCACTTTG PS/MOE miR-93 (Tuschl) 344271 1770 ACAAGATCGGATCTACGGGT PS/MOE miR-99a (Tuschl) 344272 1816 CAAACACCATTGTCACACTCCA PS/MOE miR-122a,b (Tuschl) 344273 1920 TGTCAATTCATAGGTCAG PS/MOE miR-192 (Tuschl) 344274 1832 CCAACAACATGAAACTACCTA PS/MOE miR-196 (Tuschl) 344275 1912 TCTAGTGGTCCTAAACATTTCA PS/MOE miR-203 (Tuschl) 344276 1828 CAGGCATAGGATGACAAAGGGAA PS/MOE miR-204 (Tuschl) 344277 1767 AATACATACTTCTTTACATTCCA PS/MOE miR-1d (Tuschl) 344278 1769 ACAAATTCGGATCTACAGGGTA PS/MOE 5-10-7 miR-10 (Tuschl) gapmer 344279 1774 ACACAAATTCGGTTCTACAGGG PS/MOE 5-10-7 miR-10b gapmer (Tuschl) 344280 1890 TAACCGATTTCAAATGGTGCTA PS/MOE 5-10-7 miR-29c gapmer (Tuschl) 344281 1867 GCACGAACAGCACTTTG PS/MOE 5-10-2 miR-93 (Tuschl) gapmer 344282 1770 ACAAGATCGGATCTACGGGT PS/MOE 5-10-5 miR-99a gapmer (Tuschl) 344283 1816 CAAACACCATTGTCACACTCCA PS/MOE 5-10-7 miR-122a,b gapmer (Tuschl) 344284 1920 TGTCAATTCATAGGTCAG PS/MOE 5-10-3 miR-192 gapmer (Tuschl) 344285 1832 CCAACAACATGAAACTACCTA PS/MOE 5-10-6 miR-196 gapmer (Tuschl) 344286 1912 TCTAGTGGTCCTAAACATTTCA PS/MOE 5-10-7 miR-203 gapmer (Tuschl) 344287 1828 CAGGCATAGGATGACAAAGGGAA PS/MOE 5-10-8 miR-204 gapmer (Tuschl) 344288 1767 AATACATACTTCTTTACATTCCA PS/MOE 5-10-8 miR-1d (Tuschl) gapmer 344336 1918 TGGCATTCACCGCGTGCCTTA PS/MOE mir-124a (Kosik) 344337 1754 AAAGAGACCGGTTCACTGTGA PS/MOE mir-128 (Kosik) 344338 1812 ATGCCCTTTTAACATTGCACTG PS/MOE mir-130 (Kosik) 344339 1854 CTCACCGACAGCGTTGAATGTT PS/MOE mir-178 (Kosik) 344340 1921 TGTCCGTGGTTCTACCCTGTGGTA PS/MOE mir-239* (Kosik) 344341 1823 CACATGGTTAGATCAAGCACAA PS/MOE mir-253* (Kosik) 344342 1814 ATGCTTTTTGGGGTAAGGGCTT PS/MOE mir-129as/mir- 258* (Kosik) 344343 1811 ATGCCCTTTCATCATTGCACTG PS/MOE mir-266* (Kosik) 344344 1918 TGGCATTCACCGCGTGCCTTA PS/MOE 5-10-6 mir-124a gapmer (Kosik) 344345 1754 AAAGAGACCGGTTCACTGTGA PS/MOE 5-10-6 mir-128 (Kosik) gapmer 344346 1812 ATGCCCTTTTAACATTGCACTG PS/MOE 5-10-7 mir-130 (Kosik) gapmer 344347 1854 CTCACCGACAGCGTTGAATGTT PS/MOE 5-10-7 mir-178 (Kosik) gapmer 344348 1921 TGTCCGTGGTTCTACCCTGTGGTA PS/MOE 5-10-9 mir-239* gapmer (Kosik) 344349 1823 CACATGGTTAGATCAAGCACAA PS/MOE 5-10-7 mir-253* gapmer (Kosik) 344350 1814 ATGCTTTTTGGGGTAAGGGCTT PS/MOE 5-10-7 mir-129as/mir- gapmer 258* (Kosik) 344351 1811 ATGCCCTTTCATCATTGCACTG PS/MOE 5-10-7 mir-266* gapmer (Kosik) 344611 1785 ACATTTTTCGTTATTGCTCTTGA PS/MOE mir-240* (Kosik) 344612 1790 ACGGAAGGGCAGAGAGGGCCAG PS/MOE mir-232* (Kosik) 344613 1775 ACACCAATGCCCTAGGGGATGCG PS/MOE mir-227* (Kosik) 344614 1834 CCAGCAGCACCTGGGGCAGT PS/MOE mir-226* (Kosik) 344615 1900 TCAACAAAATCACTGATGCTGGA PS/MOE mir-244* (Kosik) 344616 1800 AGAGGTCGACCGTGTAATGTGC PS/MOE mir-224* (Kosik) 344617 1862 GACGGGTGCGATTTCTGTGTGAGA PS/MOE mir-248* (Kosik) 344618 1785 ACATTTTTCGTTATTGCTCTTGA PS/MOE 5-10-8 mir-240* gapmer (Kosik) 344619 1790 ACGGAAGGGCAGAGAGGGCCAG PS/MOE 5-10-7 mir-232* gapmer (Kosik) 344620 1775 ACACCAATGCCCTAGGGGATGCG PS/MOE 5-10-8 mir-227* gapmer (Kosik) 344621 1834 CCAGCAGCACCTGGGGCAGT PS/MOE 5-10-5 mir-226* gapmer (Kosik) 344622 1900 TCAACAAAATCACTGATGCTGGA PS/MOE 5-10-8 mir-244* gapmer (Kosik) 344623 1800 AGAGGTCGACCGTGTAATGTGC PS/MOE 5-10-7 mir-224* gapmer (Kosik) 344624 1862 GACGGGTGCGATTTCTGTGTGAGA PS/MOE 5-10-9 mir-248* gapmer (Kosik) 345344 291 CTACCATAGGGTAAAACCACT PS/MOE 5-10-6 mir-140 gapmer 345345 292 GCTGCAAACATCCGACTGAAAG PS/MOE 5-10-7 mir-30a gapmer 345346 293 ACAACCAGCTAAGACACTGCCA PS/MOE 5-10-7 mir-34 gapmer 345347 294 AACACTGATTTCAAATGGTGCTA PS/MOE 5-10-8 mir-29b gapmer 345348 295 CGCCAATATTTACGTGCTGCTA PS/MOE 5-10-7 mir-16 gapmer 345350 297 AACAAAATCACTAGTCTTCCA PS/MOE 5-10-6 mir-7 gapmer 345351 299 AAAAGAGACCGGTTCACTGTGA PS/MOE 5-10-7 mir-128a gapmer 345352 300 TCACTTTTGTGACTATGCAA PS/MOE 5-10-5 mir-153 gapmer 345353 301 CAGAACTTAGCCACTGTGAA PS/MOE 5-10-5 mir-27b gapmer 345354 302 GCAAAAATGTGCTAGTGCCAAA PS/MOE 5-10-7 mir-96 gapmer 345355 303 ACTACCTGCACTGTAAGCACTTTG PS/MOE 5-10-9 mir-17as/mir-91 gapmer 345356 304 CGCGTACCAAAAGTAATAATG PS/MOE 5-10-6 mir-123/mir- gapmer 126as 345357 305 GCGACCATGGCTGTAGACTGTTA PS/MOE 5-10-8 mir-132 gapmer 345358 306 AATGCCCCTAAAAATCCTTAT PS/MOE 5-10-6 mir-108 gapmer 345359 308 AGCACAAACTACTACCTCA PS/MOE 5-10-4 let-7i gapmer 345360 309 GGCCGTGACTGGAGACTGTTA PS/MOE 5-10-6 mir-212 gapmer 345361 311 AACCACACAACCTACTACCTCA PS/MOE 5-10-7 let-7b gapmer 345362 312 ATACATACTTCTTTACATTCCA PS/MOE 5-10-7 mir-1d gapmer 345363 313 ACAAACACCATTGTCACACTCCA PS/MOE 5-10-8 mir-122a gapmer 345364 314 ACAGTTCTTCAACTGGCAGCTT PS/MOE 5-10-7 mir-22 gapmer 345365 315 ACAGGCCGGGACAAGTGCAATA PS/MOE 5-10-7 mir-92 gapmer 345366 316 GTAGTGCTTTCTACTTTATG PS/MOE 5-10-5 mir-142 gapmer 345367 317 CAGTGAATTCTACCAGTGCCATA PS/MOE 5-10-8 mir-183 gapmer 345368 318 CTGCCTGTCTGTGCCTGCTGT PS/MOE 5-10-6 mir-214 gapmer 345369 320 GGCTGTCAATTCATAGGTCAG PS/MOE 5-10-6 mir-192 gapmer 345370 321 AACTATACAACCTACTACCTCA PS/MOE 5-10-7 let-7a gapmer 345371 323 CAGACTCCGGTGGAATGAAGGA PS/MOE 5-10-7 mir-205 gapmer 345372 324 TCATAGCCCTGTACAATGCTGCT PS/MOE 5-10-8 mir-103 gapmer 345373 325 AGCCTATCCTGGATTACTTGAA PS/MOE 5-10-7 mir-26a gapmer 345374 326 CAATGCAACTACAATGCAC PS/MOE 5-10-4 mir-33a gapmer 345375 327 CCCAACAACATGAAACTACCTA PS/MOE 5-10-7 mir-196 gapmer 345376 328 TGATAGCCCTGTACAATGCTGCT PS/MOE 5-10-8 mir-107 gapmer 345377 329 GCTACCTGCACTGTAAGCACTTTT PS/MOE 5-10-9 mir-106 gapmer 345378 330 AACTATACAATCTACTACCTCA PS/MOE 5-10-7 let-7f gapmer 345379 332 GCCCTTTTAACATTGCACTG PS/MOE 5-10-5 mir-130a gapmer 345380 333 ACATGGTTAGATCAAGCACAA PS/MOE 5-10-6 mir-218 gapmer 345381 334 TGGCATTCACCGCGTGCCTTAA PS/MOE 5-10-7 mir-124a gapmer 345382 335 TCAACATCAGTCTGATAAGCTA PS/MOE 5-10-7 mir-21 gapmer 345383 336 CTAGTACATCATCTATACTGTA PS/MOE 5-10-7 mir-144 gapmer 345384 337 GAAACCCAGCAGACAATGTAGCT PS/MOE 5-10-8 mir-221 gapmer 345385 338 GAGACCCAGTAGCCAGATGTAGCT PS/MOE 5-10-9 mir-222 gapmer 345386 339 CTTCCAGTCGGGGATGTTTACA PS/MOE 5-10-7 mir-30d gapmer 345387 340 TCAGTTTTGCATGGATTTGCACA PS/MOE 5-10-8 mir-19b gapmer 345388 341 GAAAGAGACCGGTTCACTGTGA PS/MOE 5-10-7 mir-128b gapmer 345389 343 TAGCTGGTTGAAGGGGACCAA PS/MOE 5-10-6 mir-133b gapmer 345390 344 ACTATGCAACCTACTACCTCT PS/MOE 5-10-6 let-7d gapmer 345391 345 TGTAAACCATGATGTGCTGCTA PS/MOE 5-10-7 mir-15b gapmer 345392 347 GAACAGATAGTCTAAACACTGGG PS/MOE 5-10-8 mir-199b gapmer 345393 348 ACTATACAACCTCCTACCTCA PS/MOE 5-10-6 let-7e gapmer 345394 350 AGGCATAGGATGACAAAGGGAA PS/MOE 5-10-7 mir-204 gapmer 345395 351 AAGGGATTCCTGGGAAAACTGGAC PS/MOE 5-10-9 mir-145 gapmer 345396 353 CTACCTGCACTATAAGCACTTTA PS/MOE 5-10-8 mir-20 gapmer 345397 354 ACAGCTGGTTGAAGGGGACCAA PS/MOE 5-10-7 mir-133a gapmer 345398 355 GATTCACAACACCAGCT PS/MOE 5-10-2 mir-138 gapmer 345399 357 TCACAAGTTAGGGTCTCAGGGA PS/MOE 5-10-7 mir-125b gapmer 345400 358 GAACAGGTAGTCTGAACACTGGG PS/MOE 5-10-8 mir-199a gapmer 345401 359 AACCCACCGACAGCAATGAATGTT PS/MOE 5-10-9 mir-181b gapmer 345402 360 CCATCTTTACCAGACAGTGTT PS/MOE 5-10-6 mir-141 gapmer 345403 361 TATCTGCACTAGATGCACCTTA PS/MOE 5-10-7 mir-18 gapmer 345404 362 AAAGTGTCAGATACGGTGTGG PS/MOE 5-10-6 mir-220 gapmer 345405 363 CTGTTCCTGCTGAACTGAGCCA PS/MOE 5-10-7 mir-24 gapmer 345406 364 AGGCGAAGGATGACAAAGGGAA PS/MOE 5-10-7 mir-211 gapmer 345407 365 TCAGTTATCACAGTACTGTA PS/MOE 5-10-5 mir-101 gapmer 345408 366 GCTGAGTGTAGGATGTTTACA PS/MOE 5-10-6 mir-30b gapmer 345409 367 CACAAATTCGGATCTACAGGGTA PS/MOE 5-10-8 mir-10a gapmer 345410 368 TCAGTTTTGCATAGATTTGCACA PS/MOE 5-10-8 mir-19a gapmer 345411 369 CACAAACCATTATGTGCTGCTA PS/MOE 5-10-7 mir-15a gapmer 345412 370 CTACGCGTATTCTTAAGCAATA PS/MOE 5-10-7 mir-137 gapmer 345413 371 AGAATTGCGTTTGGACAATCA PS/MOE 5-10-6 mir-219 gapmer 345414 372 ACAAAGTTCTGTGATGCACTGA PS/MOE 5-10-7 mir-148b gapmer 345415 374 CACAGTTGCCAGCTGAGATTA PS/MOE 5-10-6 mir-216 gapmer 345416 375 CACAAGTTCGGATCTACGGGTT PS/MOE 5-10-7 mir-100 gapmer 345417 376 CCGGCTGCAACACAAGACACGA PS/MOE 5-10-7 mir-187 gapmer 345418 377 CAGCCGCTGTCACACGCACAG PS/MOE 5-10-6 mir-210 gapmer 345419 378 GTCTGTCAATTCATAGGTCAT PS/MOE 5-10-6 mir-215 gapmer 345420 379 GGGGTATTTGACAAACTGACA PS/MOE 5-10-6 mir-223 gapmer 345421 380 GCTGAGAGTGTAGGATGTTTACA PS/MOE 5-10-8 mir-30c gapmer 345422 381 AACCTATCCTGAATTACTTGAA PS/MOE 5-10-7 mir-26b gapmer 345423 382 CCAAGTTCTGTCATGCACTGA PS/MOE 5-10-6 mir-152 gapmer 345424 383 ATCACATAGGAATAAAAAGCCATA PS/MOE 5-10-9 mir-135 gapmer 345425 384 ATCCAATCAGTTCCTGATGCAGTA PS/MOE 5-10-9 mir-217 gapmer 345426 386 CAATGCAACAGCAATGCAC PS/MOE 5-10-4 mir-33b gapmer 345427 387 TGTGAGTTCTACCATTGCCAAA PS/MOE 5-10-7 mir-182 gapmer 345428 388 ACAAAGTTCTGTAGTGCACTGA PS/MOE 5-10-7 mir-148a gapmer 345429 389 GGAAATCCCTGGCAATGTGAT PS/MOE 5-10-6 mir-23a gapmer 345430 390 ACTCACCGACAGGTTGAATGTT PS/MOE 5-10-7 mir-181c gapmer 345431 391 ACTGTAGGAATATGTTTGATA PS/MOE 5-10-6 hypothetical gapmer miRNA-013 345432 392 ATTAAAAAGTCCTCTTGCCCA PS/MOE 5-10-6 hypothetical gapmer miRNA-023 345433 393 GCTGCCGTATATGTGATGTCA PS/MOE 5-10-6 hypothetical gapmer miRNA-030 345434 394 GGTAGGTGGAATACTATAACA PS/MOE 5-10-6 hypothetical gapmer miRNA-033 345435 395 TAAACATCACTGCAAGTCTTA PS/MOE 5-10-6 hypothetical gapmer miRNA-039 345436 396 TTGTAAGCAGTTTTGTTGACA PS/MOE 5-10-6 hypothetical gapmer miRNA-040 345437 397 TCACAGAGAAAACAACTGGTA PS/MOE 5-10-6 hypothetical gapmer miRNA-041 345438 398 CCTCTCAAAGATTTCCTGTCA PS/MOE 5-10-6 hypothetical gapmer miRNA-043 345439 399 TGTCAGATAAACAGAGTGGAA PS/MOE 5-10-6 hypothetical gapmer miRNA-044 345440 400 GAGAATCAATAGGGCATGCAA PS/MOE 5-10-6 hypothetical gapmer miRNA-055 345441 401 AAGAACATTAAGCATCTGACA PS/MOE 5-10-6 hypothetical gapmer miRNA-058 345442 402 AATCTCTGCAGGCAAATGTGA PS/MOE 5-10-6 hypothetical gapmer miRNA-070 345443 403 AAACCCCTATCACGATTAGCA PS/MOE 5-10-6 hypothetical gapmer miRNA-071 345444 404 GCCCCATTAATATTTTAACCA PS/MOE 5-10-6 hypothetical gapmer miRNA-075 345445 405 CCCAATATCAAACATATCA PS/MOE 5-10-4 hypothetical gapmer miRNA-079 345446 406 TATGATAGCTTCCCCATGTAA PS/MOE 5-10-6 hypothetical gapmer miRNA-083 345447 407 CCTCAATTATTGGAAATCACA PS/MOE 5-10-6 hypothetical gapmer miRNA-088 345448 408 ATTGATGCGCCATTTGGCCTA PS/MOE 5-10-6 hypothetical gapmer miRNA-090 345449 409 CTGTGACTTCTCTATCTGCCT PS/MOE 5-10-6 hypothetical gapmer miRNA-099 345450 410 AAACTTGTTAATTGACTGTCA PS/MOE 5-10-6 hypothetical gapmer miRNA-101 345451 411 AAAGAAGTATATGCATAGGAA PS/MOE 5-10-6 hypothetical gapmer miRNA-105 345452 412 GATAAAGCCAATAAACTGTCA PS/MOE 5-10-6 hypothetical gapmer miRNA-107 345453 413 TCCGAGTCGGAGGAGGAGGAA PS/MOE 5-10-6 hypothetical gapmer miRNA-111 345454 414 ATCATTACTGGATTGCTGTAA PS/MOE 5-10-6 hypothetical gapmer miRNA-120 345455 415 CAAAAATTATCAGCCAGTTTA PS/MOE 5-10-6 hypothetical gapmer miRNA-137 345456 416 AATCTCATTTTCATACTTGCA PS/MOE 5-10-6 hypothetical gapmer miRNA-138 345457 417 AGAAGGTGGGGAGCAGCGTCA PS/MOE 5-10-6 hypothetical gapmer miRNA-142 345458 418 CAAAATTGCAAGCAAATTGCA PS/MOE 5-10-6 hypothetical gapmer miRNA-143 345459 419 TCCACAAAGCTGAACATGTCT PS/MOE 5-10-6 hypothetical gapmer miRNA-144 345460 420 TATTATCAGCATCTGCTTGCA PS/MOE 5-10-6 hypothetical gapmer miRNA-153 345461 421 AATAACACACATCCACTTTAA PS/MOE 5-10-6 hypothetical gapmer miRNA-154 345462 422 AAGAAGGAAGGAGGGAAAGCA PS/MOE 5-10-6 hypothetical gapmer miRNA-156 345463 423 ATGACTACAAGTTTATGGCCA PS/MOE 5-10-6 hypothetical gapmer miRNA-161 345464 424 CAAAACATAAAAATCCTTGCA PS/MOE 5-10-6 hypothetical gapmer miRNA-164 345465 425 TTACAGGTGCTGCAACTGGAA PS/MOE 5-10-6 hypothetical gapmer miRNA-166 345466 426 AGCAGGTGAAGGCACCTGGCT PS/MOE 5-10-6 hypothetical gapmer miRNA-168 345467 427 TATGAAATGCCAGAGCTGCCA PS/MOE 5-10-6 hypothetical gapmer miRNA-169 345468 428 CCAAGTGTTAGAGCAAGATCA PS/MOE 5-10-6 hypothetical gapmer miRNA-170 345469 429 AACGATAAAACATACTTGTCA PS/MOE 5-10-6 hypothetical gapmer miRNA-171 345470 430 AGTAACTTCTTGCAGTTGGA PS/MOE 5-10-5 hypothetical gapmer miRNA-172 345471 431 AGCCTCCTTCTTCTCGTACTA PS/MOE 5-10-6 hypothetical gapmer miRNA-173 345472 432 ACCTCAGGTGGTTGAAGGAGA PS/MOE 5-10-6 hypothetical gapmer miRNA-175 345473 433 ATATGTCATATCAAACTCCTA PS/MOE 5-10-6 hypothetical gapmer miRNA-176 345474 434 GTGAGAGTAGCATGTTTGTCT PS/MOE 5-10-6 hypothetical gapmer miRNA-177 345475 435 TGAAGGTTCGGAGATAGGCTA PS/MOE 5-10-6 hypothetical gapmer miRNA-178 345476 436 AATTGGACAAAGTGCCTTTCA PS/MOE 5-10-6 hypothetical gapmer miRNA-179 345477 437 ACCGAACAAAGTCTGACAGGA PS/MOE 5-10-6 hypothetical gapmer miRNA-180 345478 438 AACTACTTCCAGAGCAGGTGA PS/MOE 5-10-6 hypothetical gapmer miRNA-181 345479 439 GTAAGCGCAGCTCCACAGGCT PS/MOE 5-10-6 hypothetical gapmer miRNA-183 345480 440 GAGCTGCTCAGCTGGCCATCA PS/MOE 5-10-6 hypothetical gapmer miRNA-185 345481 441 TACTTTTCATTCCCCTCACCA PS/MOE 5-10-6 hypothetical gapmer miRNA-188 345482 236 TAGCTTATCAGACTGATGTTGA PS/MOE 5-10-7 miR-104 gapmer (Mourelatos) 345483 1780 ACAGGAGTCTGAGCATTTGA PS/MOE 5-10-5 miR-105 gapmer (Mourelatos) 345484 1882 GGAACTTAGCCACTGTGAA PS/MOE 5-10-4 miR-27 gapmer (Mourelatos) 345485 848 CTACCTGCACGAACAGCACTTT PS/MOE 5-10-7 miR-93 gapmer (Mourelatos) 345486 855 TGCTCAATAAATACCCGTTGAA PS/MOE 5-10-7 miR-95 gapmer (Mourelatos) 345487 1821 CACAAGATCGGATCTACGGGTT PS/MOE 5-10-7 miR-99 gapmer (Mourelatos) 345488 1903 TCAGACCGAGACAAGTGCAATG PS/MOE 5-10-7 miR-25 (Tuschl) gapmer 345489 1853 CTCAATAGACTGTGAGCTCCTT PS/MOE 5-10-7 miR-28 (Tuschl) gapmer 345490 1825 CAGCTATGCCAGCATCTTGCC PS/MOE 5-10-6 miR-31 (Tuschl) gapmer 345491 1865 GCAACTTAGTAATGTGCAATA PS/MOE 5-10-6 miR-32 (Tuschl) gapmer 345492 1897 TAGGAGAGAGAAAAAGACTGA PS/MOE 5-10-6 miR-14 gapmer 345493 854 GGAGTGAAGACACGGAGCCAGA PS/MOE 5-10-7 miR-149 gapmer 345494 1845 CGCAAGGTCGGTTCTACGGGTG PS/MOE 5-10-7 miR-99b gapmer 345495 852 CACAGGTTAAAGGGTCTCAGGGA PS/MOE 5-10-8 miR-125a gapmer 345496 853 AGCCAAGCTCAGACGGATCCGA PS/MOE 5-10-7 miR-127 gapmer 345497 1909 TCCATCATCAAAACAAATGGAGT PS/MOE 5-10-8 miR-136 gapmer 345498 1843 CGAAGGCAACACGGATAACCTA PS/MOE 5-10-7 miR-154 gapmer 345499 1880 GCTTCCAGTCGAGGATGTTTACA PS/MOE 5-10-8 miR-30a-s gapmer 345500 1911 TCCGTGGTTCTACCCTGTGGTA PS/MOE 5-10-7 miR-140-as gapmer 345501 1836 CCATAAAGTAGGAAACACTACA PS/MOE 5-10-7 miR-142-as gapmer 345502 1761 AACAGGTAGTCTGAACACTGGG PS/MOE 5-10-7 miR-199-s gapmer 345503 1762 AACCAATGTGCAGACTACTGTA PS/MOE 5-10-7 miR-199-as gapmer 345504 1904 TCATACAGCTAGATAACCAAAGA PS/MOE 5-10-8 miR-9 gapmer 345505 1773 ACAAGTGCCTTCACTGCAGT PS/MOE 5-10-5 miR-17 gapmer 345506 1871 GCATTATTACTCACGGTACGA PS/MOE 5-10-6 miR-126a gapmer 345507 1787 ACCTAATATATCAAACATATCA PS/MOE 5-10-7 miR-190 gapmer 345508 1766 AAGCCCAAAAGGAGAATTCTTTG PS/MOE 5-10-8 miR-186 gapmer 345509 1839 CCTATCTCCCCTCTGGACC PS/MOE 5-10-4 miR-198a gapmer 345510 1806 AGCTGCTTTTGGGATTCCGTTG PS/MOE 5-10-7 miR-191c gapmer 345511 760 CCACACACTTCCTTACATTCCA PS/MOE 5-10-7 miR-206d gapmer 345512 761 ATCTGCACTGTCAGCACTTT PS/MOE 5-10-5 miR-94 gapmer 345513 762 ACCCTTATCAGTTCTCCGTCCA PS/MOE 5-10-7 miR-184 gapmer 345514 763 GCCAATATTTCTGTGCTGCTA PS/MOE 5-10-6 miR-195 gapmer 345515 764 CTGGGACTTTGTAGGCCAGTT PS/MOE 5-10-6 miR-193 gapmer 345516 1861 GAACTGCCTTTCTCTCCA PS/MOE 5-10-3 miR-185 gapmer 345517 1786 ACCCTCCACCATGCAAGGGATG PS/MOE 5-10-7 miR-188 gapmer 345518 1879 GCTGGGTGGAGAAGGTGGTGAA PS/MOE 5-10-7 miR-197a gapmer 345519 1906 TCCACATGGAGTTGCTGTTACA PS/MOE 5-10-7 miR-194 gapmer 345520 1771 ACAAGCTTTTTGCTCGTCTTAT PS/MOE 5-10-7 miR-208 gapmer 345521 938 AGACACGTGCACTGTAGA PS/MOE 5-10-3 miR-139 gapmer 345522 1887 GTCATCATTACCAGGCAGTATTA PS/MOE 5-10-8 miR-200b gapmer 345523 1831 CATCGTTACCAGACAGTGTTA PS/MOE 5-10-6 miR-200a gapmer 345524 1827 CAGCTTTCAAAATGATCTCAC PS/MOE 5-10-6 miR-Bantam gapmer 345922 1783 ACAGTGCTTCATCTCA PO/6MOE-10deoxy mir-143 hemimer 345923 1848 CTACAGTGCTTCATCTC PO/6MOE-11deoxy mir-143 hemimer 345924 1876 GCTACAGTGCTTCATCT PO/6MOE-11deoxy mir-143 hemimer 345925 1875 GCTACAGTGCTTCATC PO/6MOE-10deoxy mir-143 hemimer 345926 1803 AGCTACAGTGCTTCAT PO/6MOE-10deoxy mir-143 hemimer 345927 1863 GAGCTACAGTGCTTCA PO/6MOE-10deoxy mir-143 hemimer 345928 1916 TGAGCTACAGTGCTTC PO/6MOE-10deoxy mir-143 hemimer 346685 1884 GGCGGAACTTAGCCACTGTGAA PS/MOE miR-27a (RFAM- Human) 346686 1857 CTTCAGTTATCACAGTACTGTA PS/MOE miR-101 (RFAM- Human) 346687 1802 AGCAAGCCCAGACCGCAAAAAG PS/MOE miR-129b (RFAM- Human) 346688 1898 TAGTTGGCAAGTCTAGAACCA PS/MOE miR-182* (RFAM- Human) 346689 1830 CATCATTACCAGGCAGTATTAGAG PS/MOE miR-200a (RFAM- Human) 346690 1792 ACTGATATCAGCTCAGTAGGCAC PS/MOE miR-189 (RFAM- Human) 346691 1870 GCAGAAGCATTTCCACACAC PS/MOE miR-147 (RFAM- Human) 346692 1889 TAAACGGAACCACTAGTGACTTG PS/MOE miR-224 (RFAM- Human) 346693 1838 CCCTCTGGTCAACCAGTCACA PS/MOE miR-134 (RFAM- Human) 346694 1763 AACCCATGGAATTCAGTTCTCA PS/MOE miR-146 (RFAM- Human) 346695 1824 CACTGGTACAAGGGTTGGGAGA PS/MOE miR-150 (RFAM- Human) 346696 1893 TACCTGCACTATAAGCACTTTA PS/MOE mir-20 346697 1788 ACCTATCCTGAATTACTTGAA PS/MOE mir-26b 346698 1793 ACTGATTTCAAATGGTGCTA PS/MOE mir-29b 346699 1847 CGGCTGCAACACAAGACACGA PS/MOE miR-187 (RFAM- Human) 346700 1844 CGACCATGGCTGTAGACTGTTA PS/MOE miR-132 (RFAM- Human) 346701 1901 TCACATAGGAATAAAAAGCCATA PS/MOE miR-135 (RFAM- Human) 346702 1893 TACCTGCACTATAAGCACTTTA PS/MOE 5-10-7 mir-20 gapmer 346703 1788 ACCTATCCTGAATTACTTGAA PS/MOE 5-10-6 mir-26b gapmer 346704 1884 GGCGGAACTTAGCCACTGTGAA PS/MOE 5-10-7 miR-27a (RFAM- gapmer Human) 346705 1857 CTTCAGTTATCACAGTACTGTA PS/MOE 5-10-7 miR-101 (RFAM- gapmer Human) 346706 1793 ACTGATTTCAAATGGTGCTA PS/MOE 5-10-5 mir-29b gapmer 346707 1847 CGGCTGCAACACAAGACACGA PS/MOE 5-10-6 miR-187 (RFAM- gapmer Human) 346708 1844 CGACCATGGCTGTAGACTGTTA PS/MOE 5-10-7 miR-132 (RFAM- gapmer Human) 346709 1901 TCACATAGGAATAAAAAGCCATA PS/MOE 5-10-8 miR-135 (RFAM- gapmer Human) 346710 1802 AGCAAGCCCAGACCGCAAAAAG PS/MOE 5-10-7 miR-129b (RFAM- gapmer Human) 346711 1898 TAGTTGGCAAGTCTAGAACCA PS/MOE 5-10-6 miR-182* (RFAM- gapmer Human) 346712 1830 CATCATTACCAGGCAGTATTAGAG PS/MOE 5-10-9 miR-200a (RFAM- gapmer Human) 346713 1792 ACTGATATCAGCTCAGTAGGCAC PS/MOE 5-10-8 miR-189 (RFAM- gapmer Human) 346714 1870 GCAGAAGCATTTCCACACAC PS/MOE 5-10-5 miR-147 (RFAM- gapmer Human) 346715 1889 TAAACGGAACCACTAGTGACTTG PS/MOE 5-10-8 miR-224 (RFAM- gapmer Human) 346716 1838 CCCTCTGGTCAACCAGTCACA PS/MOE 5-10-6 miR-134 (RFAM- gapmer Human) 346717 1763 AACCCATGGAATTCAGTTCTCA PS/MOE 5-10-7 miR-146 (RFAM- gapmer Human) 346718 1824 CACTGGTACAAGGGTTGGGAGA PS/MOE 5-10-7 miR-150 (RFAM- gapmer Human) 346905 1907 TCCAGTCAAGGATGTTTACA PS/MOE miR-30e (RFAM- M. musculus) 346906 1781 ACAGGATTGAGGGGGGGCCCT PS/MOE miR-296 (RFAM- M. musculus) 346907 1815 ATGTATGTGGGACGGTAAACCA PS/MOE miR-299 (RFAM- M. musculus) 346908 1881 GCTTTGACAATACTATTGCACTG PS/MOE miR-301 (RFAM- M. musculus) 346909 1902 TCACCAAAACATGGAAGCACTTA PS/MOE miR-302 (RFAM- M. musculus) 346910 1866 GCAATCAGCTAACTACACTGCCT PS/MOE miR-34a (RFAM- M. musculus) 346911 1776 ACACTGATTTCAAATGGTGCTA PS/MOE miR-29b (RFAM- M. musculus) 346912 1851 CTAGTGGTCCTAAACATTTCA PS/MOE miR-203 (RFAM- M. musculus) 346913 1795 AGAAAGGCAGCAGGTCGTATAG PS/MOE let-7d* (RFAM- M. musculus) 346914 1810 ATCTGCACTGTCAGCACTTTA PS/MOE miR-106b (RFAM- M. musculus) 346915 1784 ACATCGTTACCAGACAGTGTTA PS/MOE miR-200a (RFAM- M. musculus) 346916 1874 GCGGAACTTAGCCACTGTGAA PS/MOE miR-27a (RFAM- M. musculus) 346917 1826 CAGCTATGCCAGCATCTTGCCT PS/MOE miR-31 (RFAM-M. musculus) 346918 1829 CAGGCCGGGACAAGTGCAATA PS/MOE miR-92 (RFAM-M. musculus) 346919 1849 CTACCTGCACGAACAGCACTTTG PS/MOE miR-93 (RFAM-M. musculus) 346920 1801 AGCAAAAATGTGCTAGTGCCAAA PS/MOE miR-96 (RFAM-M. musculus) 346921 1759 AACAACCAGCTAAGACACTGCCA PS/MOE miR-172 (RFAM- M. musculus) 346922 1907 TCCAGTCAAGGATGTTTACA PS/MOE 5-10-5 miR-30e (RFAM- gapmer M. musculus) 346923 1781 ACAGGATTGAGGGGGGGCCCT PS/MOE 5-10-6 miR-296 (RFAM- gapmer M. musculus) 346924 1815 ATGTATGTGGGACGGTAAACCA PS/MOE 5-10-7 miR-299 (RFAM- gapmer M. musculus) 346925 1881 GCTTTGACAATACTATTGCACTG PS/MOE 5-10-8 miR-301 (RFAM- gapmer M. musculus) 346926 1902 TCACCAAAACATGGAAGCACTTA PS/MOE 5-10-8 miR-302 (RFAM- gapmer M. musculus) 346927 1866 GCAATCAGCTAACTACACTGCCT PS/MOE 5-10-8 miR-34a (RFAM- gapmer M. musculus) 346928 1776 ACACTGATTTCAAATGGTGCTA PS/MOE 5-10-7 miR-29b (RFAM- gapmer M. musculus) 346929 1851 CTAGTGGTCCTAAACATTTCA PS/MOE 5-10-6 miR-203 (RFAM- gapmer M. musculus) 346930 1795 AGAAAGGCAGCAGGTCGTATAG PS/MOE 5-10-7 let-7d* (RFAM- gapmer M. musculus) 346931 1810 ATCTGCACTGTCAGCACTTTA PS/MOE 5-10-6 miR-106b (RFAM- gapmer M. musculus) 346932 1784 ACATCGTTACCAGACAGTGTTA PS/MOE 5-10-7 miR-200a (RFAM- gapmer M. musculus) 346933 1874 GCGGAACTTAGCCACTGTGAA PS/MOE 5-10-6 miR-27a (RFAM- gapmer M. musculus) 346934 1826 CAGCTATGCCAGCATCTTGCCT PS/MOE 5-10-7 miR-31 (RFAM-M. musculus) gapmer 346935 1829 CAGGCCGGGACAAGTGCAATA PS/MOE 5-10-6 miR-92 (RFAM-M. musculus) gapmer 346936 1849 CTACCTGCACGAACAGCACTTTG PS/MOE 5-10-8 miR-93 (RFAM-M. musculus) gapmer 346937 1801 AGCAAAAATGTGCTAGTGCCAAA PS/MOE 5-10-8 miR-96 (RFAM-M. musculus) gapmer 346938 1759 AACAACCAGCTAAGACACTGCCA PS/MOE 5-10-8 miR-172 (RFAM- gapmer M. musculus) 347385 1782 ACAGTGCTTCATCTC PO/6MOE-9deoxy mir-143 hemimer 347386 1848 CTACAGTGCTTCATCTC PO/6MOE-11deoxy mir-143 hemimer 347387 1876 GCTACAGTGCTTCATCT PO/6MOE-11deoxy mir-143 hemimer 347388 1875 GCTACAGTGCTTCATC PO/6MOE-10deoxy mir-143 hemimer 347389 1803 AGCTACAGTGCTTCAT PO/6MOE-10deoxy mir-143 hemimer 347390 1863 GAGCTACAGTGCTTCA PO/6MOE-10deoxy mir-143 hemimer 347391 1916 TGAGCTACAGTGCTTC PO/6MOE-10deoxy mir-143 hemimer 347452 1783 ACAGTGCTTCATCTCA PO/6MOE-10deoxy mir-143 hemimer 347453 1783 ACAGTGCTTCATCTCA PO/6MOE-10deoxy mir-143 hemimer 348116 1922 TTCGCCCTCTCAACCCAGCTTTT PS/MOE miR-320 348117 1860 GAACCCACAATCCCTGGCTTA PS/MOE miR-321-1 348118 1886 GTAAACCATGATGTGCTGCTA PS/MOE miR-15b (Michael et al) 348119 1908 TCCATAAAGTAGGAAACACTACA PS/MOE miR-142as (Michael et al) 348120 1864 GAGCTACAGTGCTTCATCTCA PS/MOE miR-143 (Michael et al) 348121 1883 GGATTCCTGGGAAAACTGGAC PS/MOE miR-145 (Michael et al) 348122 1905 TCATCATTACCAGGCAGTATTA PS/MOE miR-200b (Michael et al) 348123 1791 ACTATACAATCTACTACCTCA PS/MOE let-7f (Michael et al) 348124 1820 CACAAATTCGGTTCTACAGGGTA PS/MOE miR-10b (Michael et al) 348125 1878 GCTGGATGCAAACCTGCAAAACT PS/MOE miR-19b (Michael et al) 348126 1873 GCCTATCCTGGATTACTTGAA PS/MOE miR-26a (Michael et al) 348127 1869 GCAGAACTTAGCCACTGTGAA PS/MOE miR-27* (Michael et al) 348128 1858 CTTCCAGTCAAGGATGTTTACA PS/MOE miR-97 (Michael et al) 348129 1855 CTGGCTGTCAATTCATAGGTCA PS/MOE miR-192 (Michael et al) 348130 1922 TTCGCCCTCTCAACCCAGCTTTT PS/MOE 5-10-8 miR-320 gapmer 348131 1860 GAACCCACAATCCCTGGCTTA PS/MOE 5-10-6 miR-321-1 gapmer 348132 1886 GTAAACCATGATGTGCTGCTA PS/MOE 5-10-6 miR-15b gapmer (Michael et al) 348133 1908 TCCATAAAGTAGGAAACACTACA PS/MOE 5-10-8 miR-142as gapmer (Michael et al) 348134 1864 GAGCTACAGTGCTTCATCTCA PS/MOE 5-10-6 miR-143 gapmer (Michael et al) 348135 1883 GGATTCCTGGGAAAACTGGAC PS/MOE 5-10-6 miR-145 gapmer (Michael et al) 348136 1905 TCATCATTACCAGGCAGTATTA PS/MOE 5-10-7 miR-200b gapmer (Michael et al) 348137 1791 ACTATACAATCTACTACCTCA PS/MOE 5-10-6 let-7f (Michael gapmer et al) 348138 1820 CACAAATTCGGTTCTACAGGGTA PS/MOE 5-10-8 miR-10b gapmer (Michael et al) 348139 1878 GCTGGATGCAAACCTGCAAAACT PS/MOE 5-10-8 miR-19b gapmer (Michael et al) 348140 1873 GCCTATCCTGGATTACTTGAA PS/MOE 5-10-6 miR-26a gapmer (Michael et al) 348141 1869 GCAGAACTTAGCCACTGTGAA PS/MOE 5-10-6 miR-27* gapmer (Michael et al) 348142 1858 CTTCCAGTCAAGGATGTTTACA PS/MOE 5-10-7 miR-97 (Michael gapmer et al) 348143 1855 CTGGCTGTCAATTCATAGGTCA PS/MOE 5-10-7 miR-192 gapmer (Michael et al) 354040 1751 AAACCACACAACCTACTACCTCA PS/MOE let-7b-Ruvkun 354041 1752 AAACCATACAACCTACTACCTCA PS/MOE let-7c-Ruvkun 354042 1764 AACTATGCAACCTACTACCTCT PS/MOE let-7d-Ruvkun 354043 1765 AACTGTACAAACTACTACCTCA PS/MOE let-7gL-Ruvkun 354044 1760 AACAGCACAAACTACTACCTCA PS/MOE let-7i-Ruvkun 354045 1924 TTGGCATTCACCGCGTGCCTTAA PS/MOE mir-124a-Ruvkun 354046 1833 CCAAGCTCAGACGGATCCGA PS/MOE mir-127-Ruvkun 354047 1896 TACTTTCGGTTATCTAGCTTTA PS/MOE mir-131-Ruvkun 354048 1846 CGGCCTGATTCACAACACCAGCT PS/MOE mir-138-Ruvkun 354049 1768 ACAAACCATTATGTGCTGCTA PS/MOE mir-15-Ruvkun 354050 1789 ACGCCAATATTTACGTGCTGCTA PS/MOE mir-16-Ruvkun 354051 1852 CTATCTGCACTAGATGCACCTTA PS/MOE mir-18-Ruvkun 354052 1779 ACAGCTGCTTTTGGGATTCCGTTG PS/MOE mir-191-Ruvkun 354053 1891 TAACCGATTTCAGATGGTGCTA PS/MOE mir-29a-Ruvkun 354054 1813 ATGCTTTGACAATACTATTGCACTG PS/MOE mir-301-Ruvkun 354055 1805 AGCTGAGTGTAGGATGTTTACA PS/MOE mir-30b-Ruvkun 354056 1804 AGCTGAGAGTGTAGGATGTTTACA PS/MOE mir-30c-Ruvkun 354057 1807 AGCTTCCAGTCGGGGATGTTTACA PS/MOE mir-30d-Ruvkun 354058 1835 CCAGCAGCACCTGGGGCAGTGG PS/MOE mir-324-3p- Ruvkun 354059 1899 TATGGCAGACTGTGATTTGTTG PS/MOE mir-7-1*-Ruvkun 354060 1850 CTACCTGCACTGTAAGCACTTTG PS/MOE mir-91-Ruvkun 354061 1822 CACATAGGAATGAAAAGCCATA PS/MOE mir-135b (Ruvkun) 354062 1895 TACTAGACTGTGAGCTCCTCGA PS/MOE mir-151* (Ruvkun) 354063 1885 GGCTATAAAGTAACTGAGACGGA PS/MOE mir-340 (Ruvkun) 354064 1923 TTCTAGGATAGGCCCAGGGGC PS/MOE mir-331 (Ruvkun) 354065 1892 TACATACTTCTTTACATTCCA PS/MOE miR-1 (RFAM) 354066 1817 CAATCAGCTAACTACACTGCCT PS/MOE miR-34c (RFAM) 354067 1837 CCCCTATCACGATTAGCATTAA PS/MOE miR-155 (RFAM) 354068 1910 TCCATCATTACCCGGCAGTATT PS/MOE miR-200c (RFAM) 354069 1818 CAATCAGCTAATGACACTGCCT PS/MOE miR-34b (RFAM) 354070 1753 AAACCCAGCAGACAATGTAGCT PS/MOE mir-221 (RFAM- M. musculus) 354071 1796 AGACCCAGTAGCCAGATGTAGCT PS/MOE mir-222 (RFAM- M. musculus) 354072 1917 TGAGCTCCTGGAGGACAGGGA PS/MOE mir-339-1 (RFAM) 354073 1925 TTTAAGTGCTCATAATGCAGT PS/MOE miR-20* (human) 354074 1926 TTTTCCCATGCCCTATACCTCT PS/MOE miR-202 (human) 354075 1856 CTTCAGCTATCACAGTACTGTA PS/MOE miR-101b 354076 1894 TACCTGCACTGTTAGCACTTTG PS/MOE miR-106a 354077 1772 ACAAGTGCCCTCACTGCAGT PS/MOE miR-17-3p 354078 1859 GAACAGGTAGTCTAAACACTGGG PS/MOE miR-199b (mouse) 354079 1915 TCTTCCCATGCGCTATACCTCT PS/MOE miR-202 (mouse) 354080 1808 AGGCAAAGGATGACAAAGGGAA PS/MOE miR-211 (mouse) 354081 1809 ATCCAGTCAGTTCCTGATGCAGTA PS/MOE miR-217 (mouse) 354082 1888 TAAACGGAACCACTAGTGACTTA PS/MOE miR-224 (RFAM mouse) 354083 1758 AACAAAATCACAAGTCTTCCA PS/MOE miR-7b 354084 1919 TGTAAGTGCTCGTAATGCAGT PS/MOE miR-20* (mouse) 354085 1778 ACACTTACTGGACACCTACTAGG PS/MOE mir-325 (human) 354086 1777 ACACTTACTGAGCACCTACTAGG PS/MOE mir-325 (mouse) 354087 1877 GCTGGAGGAAGGGCCCAGAGG PS/MOE mir-326 (human) 354088 1794 ACTGGAGGAAGGGCCCAGAGG PS/MOE mir-326 (mouse) 354089 1755 AAAGAGGTTAACCAGGTGTGTT PS/MOE mir-329-1 (human) 354090 1750 AAAAAGGTTAGCTGGGTGTGTT PS/MOE mir-329-1 (mouse) 354091 1914 TCTCTGCAGGCCGTGTGCTTTGC PS/MOE mir-330 (human) 354092 1913 TCTCTGCAGGCCCTGTGCTTTGC PS/MOE mir-330 (mouse) 354093 1757 AAAGGCATCATATAGGAGCTGGA PS/MOE mir-337 (human) 354094 1756 AAAGGCATCATATAGGAGCTGAA PS/MOE mir-337 (mouse) 354095 1872 GCCCTGGACTAGGAGTCAGCA PS/MOE mir-345 (human) 354096 1868 GCACTGGACTAGGGGTCAGCA PS/MOE mir-345 (mouse) 354097 1799 AGAGGCAGGCATGCGGGCAGACA PS/MOE mir-346 (human) 354098 1798 AGAGGCAGGCACTCGGGCAGACA PS/MOE mir-346 (mouse) 354099 1840 CCTCAAGGAGCCTCAGTCTAG PS/MOE miR-151 (mouse) 354100 1841 CCTCAAGGAGCCTCAGTCTAGT PS/MOE miR-151 (rat) 354101 1797 AGAGGCAGGCACTCAGGCAGACA PS/MOE miR-346 (rat) 354102 1819 CAATCAGCTAATTACACTGCCTA PS/MOE miR-34b (mouse) 354103 1842 CCTCAAGGAGCTTCAGTCTAGT PS/MOE miR-151 (hum) 354104 1751 AAACCACACAACCTACTACCTCA PS/MOE 5-10-8 let-7b-Ruvkun gapmer 354105 1752 AAACCATACAACCTACTACCTCA PS/MOE 5-10-8 let-7c-Ruvkun gapmer 354106 1764 AACTATGCAACCTACTACCTCT PS/MOE 5-10-7 let-7d-Ruvkun gapmer 354107 1765 AACTGTACAAACTACTACCTCA PS/MOE 5-10-7 let-7gL-Ruvkun gapmer 354108 1760 AACAGCACAAACTACTACCTCA PS/MOE 5-10-7 let-7i-Ruvkun gapmer 354109 1924 TTGGCATTCACCGCGTGCCTTAA PS/MOE 5-10-8 mir-124a-Ruvkun gapmer 354110 1833 CCAAGCTCAGACGGATCCGA PS/MOE 5-10-5 mir-127-Ruvkun gapmer 354111 1896 TACTTTCGGTTATCTAGCTTTA PS/MOE 5-10-7 mir-131-Ruvkun gapmer 354112 1846 CGGCCTGATTCACAACACCAGCT PS/MOE 5-10-8 mir-138-Ruvkun gapmer 354113 1768 ACAAACCATTATGTGCTGCTA PS/MOE 5-10-6 mir-15-Ruvkun gapmer 354114 1789 ACGCCAATATTTACGTGCTGCTA PS/MOE 5-10-8 mir-16-Ruvkun gapmer 354115 1852 CTATCTGCACTAGATGCACCTTA PS/MOE 5-10-8 mir-18-Ruvkun gapmer 354116 1779 ACAGCTGCTTTTGGGATTCCGTTG PS/MOE 5-10-9 mir-191-Ruvkun gapmer 354117 1891 TAACCGATTTCAGATGGTGCTA PS/MOE 5-10-7 mir-29a-Ruvkun gapmer 354118 1813 ATGCTTTGACAATACTATTGCACTG PS/MOE 5-10-10 mir-301-Ruvkun gapmer 354119 1805 AGCTGAGTGTAGGATGTTTACA PS/MOE 5-10-7 mir-30b-Ruvkun gapmer 354120 1804 AGCTGAGAGTGTAGGATGTTTACA PS/MOE 5-10-9 mir-30c-Ruvkun gapmer 354121 1807 AGCTTCCAGTCGGGGATGTTTACA PS/MOE 5-10-9 mir-30d-Ruvkun gapmer 354122 1835 CCAGCAGCACCTGGGGCAGTGG PS/MOE 5-10-7 mir-324-3p- gapmer Ruvkun 354123 1899 TATGGCAGACTGTGATTTGTTG PS/MOE 5-10-7 mir-7-1*-Ruvkun gapmer 354124 1850 CTACCTGCACTGTAAGCACTTTG PS/MOE 5-10-8 mir-91-Ruvkun gapmer 354125 1822 CACATAGGAATGAAAAGCCATA PS/MOE 5-10-7 mir-135b gapmer (Ruvkun) 354126 1895 TACTAGACTGTGAGCTCCTCGA PS/MOE 5-10-7 mir-151* gapmer (Ruvkun) 354127 1885 GGCTATAAAGTAACTGAGACGGA PS/MOE 5-10-8 mir-340 gapmer (Ruvkun) 354128 1923 TTCTAGGATAGGCCCAGGGGC PS/MOE 5-10-6 mir-331 gapmer (Ruvkun) 354129 1892 TACATACTTCTTTACATTCCA PS/MOE 5-10-6 miR-1 (RFAM) gapmer 354130 1817 CAATCAGCTAACTACACTGCCT PS/MOE 5-10-7 miR-34c (RFAM) gapmer 354131 1837 CCCCTATCACGATTAGCATTAA PS/MOE 5-10-7 miR-155 (RFAM) gapmer 354132 1910 TCCATCATTACCCGGCAGTATT PS/MOE 5-10-7 miR-200c (RFAM) gapmer 354133 1818 CAATCAGCTAATGACACTGCCT PS/MOE 5-10-7 miR-34b (RFAM) gapmer 354134 1753 AAACCCAGCAGACAATGTAGCT PS/MOE 5-10-7 mir-221 (RFAM- gapmer M. musculus) 354135 1796 AGACCCAGTAGCCAGATGTAGCT PS/MOE 5-10-8 mir-222 (RFAM- gapmer M. musculus) 354136 1917 TGAGCTCCTGGAGGACAGGGA PS/MOE 5-10-6 mir-339-1 gapmer (RFAM) 354137 1925 TTTAAGTGCTCATAATGCAGT PS/MOE 5-10-6 miR-20* (human) gapmer 354138 1926 TTTTCCCATGCCCTATACCTCT PS/MOE 5-10-7 miR-202 (human) gapmer 354139 1856 CTTCAGCTATCACAGTACTGTA PS/MOE 5-10-7 miR-101b gapmer 354140 1894 TACCTGCACTGTTAGCACTTTG PS/MOE 5-10-7 miR-106a gapmer 354141 1772 ACAAGTGCCCTCACTGCAGT PS/MOE 5-10-5 miR-17-3p gapmer 354142 1859 GAACAGGTAGTCTAAACACTGGG PS/MOE 5-10-8 miR-199b gapmer (mouse) 354143 1915 TCTTCCCATGCGCTATACCTCT PS/MOE 5-10-7 miR-202 (mouse) gapmer 354144 1808 AGGCAAAGGATGACAAAGGGAA PS/MOE 5-10-7 miR-211 (mouse) gapmer 354145 1809 ATCCAGTCAGTTCCTGATGCAGTA PS/MOE 5-10-9 miR-217 (mouse) gapmer 354146 1888 TAAACGGAACCACTAGTGACTTA PS/MOE 5-10-8 miR-224 (RFAM gapmer mouse) 354147 1758 AACAAAATCACAAGTCTTCCA PS/MOE 5-10-6 miR-7b gapmer 354148 1919 TGTAAGTGCTCGTAATGCAGT PS/MOE 5-10-6 miR-20* (mouse) gapmer 354149 1778 ACACTTACTGGACACCTACTAGG PS/MOE 5-10-8 mir-325 (human) gapmer 354150 1777 ACACTTACTGAGCACCTACTAGG PS/MOE 5-10-8 mir-325 (mouse) gapmer 354151 1877 GCTGGAGGAAGGGCCCAGAGG PS/MOE 5-10-6 mir-326 (human) gapmer 354152 1794 ACTGGAGGAAGGGCCCAGAGG PS/MOE 5-10-6 mir-326 (mouse) gapmer 354153 1755 AAAGAGGTTAACCAGGTGTGTT PS/MOE 5-10-7 mir-329-1 gapmer (human) 354154 1750 AAAAAGGTTAGCTGGGTGTGTT PS/MOE 5-10-7 mir-329-1 gapmer (mouse) 354155 1914 TCTCTGCAGGCCGTGTGCTTTGC PS/MOE 5-10-8 mir-330 (human) gapmer 354156 1913 TCTCTGCAGGCCCTGTGCTTTGC PS/MOE 5-10-8 mir-330 (mouse) gapmer 354157 1757 AAAGGCATCATATAGGAGCTGGA PS/MOE 5-10-8 mir-337 (human) gapmer 354158 1756 AAAGGCATCATATAGGAGCTGAA PS/MOE 5-10-8 mir-337 (mouse) gapmer 354159 1872 GCCCTGGACTAGGAGTCAGCA PS/MOE 5-10-6 mir-345 (human) gapmer 354160 1868 GCACTGGACTAGGGGTCAGCA PS/MOE 5-10-6 mir-345 (mouse) gapmer 354161 1799 AGAGGCAGGCATGCGGGCAGACA PS/MOE 5-10-8 mir-346 (human) gapmer 354162 1798 AGAGGCAGGCACTCGGGCAGACA PS/MOE 5-10-8 mir-346 (mouse) gapmer 354163 1840 CCTCAAGGAGCCTCAGTCTAG PS/MOE 5-10-6 miR-151 (mouse) gapmer 354164 1841 CCTCAAGGAGCCTCAGTCTAGT PS/MOE 5-10-7 miR-151 (rat) gapmer 354165 1797 AGAGGCAGGCACTCAGGCAGACA PS/MOE 5-10-8 miR-346 (rat) gapmer 354166 1819 CAATCAGCTAATTACACTGCCTA PS/MOE 5-10-8 miR-34b (mouse) gapmer 354167 1842 CCTCAAGGAGCTTCAGTCTAGT PS/MOE 5-10-7 miR-151 (human) gapmer

In accordance with the present invention, oligomeric compounds were designed to mimic one or more miRNAs, pre-miRNAs or pri-miRNAs. The oligomeric compounds of the present invention can also be designed to mimic a pri-miRNA, a pre-miRNA or a single- or double-stranded miRNA while incorporating certain chemical modifications that alter one or more properties of the mimic, thus creating a construct with superior properties over the endogenous pri-miRNA, pre-miRNA or miRNA. Oligomeric compounds representing synthesized miRNAs or chemically modified miRNA mimics were given internal numerical identifiers (ISIS Numbers) and are shown in Table 66. These oligomeric compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels or for their effect on downstream target RNA transcripts by quantitative real-time PCR or they can be used in other assays to investigate the role of miRNAs or miRNA downstream targets. In Table 66, “pri-miRNA” indicates the particular pri-miRNA from which the mature miRNA is normally processed when it occurs in the cellular environment. All compounds listed in Table 66 are ribonucleotides. The miRNA mimics consist of phosphorothioate internucleoside linkages, indicated by “PS” in the “Chemistry” column of Table 66, whereas synthesized miRNA oligomeric compounds with phosphodiester internucleoside linkages are indicated by “PO.”

TABLE 66 miRNAs and miRNA mimics SEQ ID Linkage ISIS # NO sequence chemistry Pri-miRNA 343092 437 ACCGAACAAAGTCTGACAGGA PO hypothetical miRNA-180 343098 1780 ACAGGAGTCTGAGCATTTGA PO miR-105 (Mourelatos) 343099 1882 GGAACTTAGCCACTGTGAA PO miR-27 (Mourelatos) 343101 855 TGCTCAATAAATACCCGTTGAA PO miR-95 (Mourelatos) 343102 1821 CACAAGATCGGATCTACGGGTT PO miR-99 (Mourelatos) 343103 1903 TCAGACCGAGACAAGTGCAATG PO miR-25 (Tuschl) 343104 1853 CTCAATAGACTGTGAGCTCCTT PO miR-28 (Tuschl) 343105 1825 CAGCTATGCCAGCATCTTGCC PO miR-31 (Tuschl) 343106 1865 GCAACTTAGTAATGTGCAATA PO miR-32 (Tuschl) 343107 854 GGAGTGAAGACACGGAGCCAGA PO miR-149 343108 1845 CGCAAGGTCGGTTCTACGGGTG PO miR-99b 343109 852 CACAGGTTAAAGGGTCTCAGGGA PO miR-125a 343110 853 AGCCAAGCTCAGACGGATCCGA PO miR-127 343111 1909 TCCATCATCAAAACAAATGGAGT PO miR-136 343112 1843 CGAAGGCAACACGGATAACCTA PO miR-154 343113 1880 GCTTCCAGTCGAGGATGTTTACA PO miR-30a-s 343114 1911 TCCGTGGTTCTACCCTGTGGTA PO miR-140-as 343115 1836 CCATAAAGTAGGAAACACTACA PO miR-142-as 343117 1762 AACCAATGTGCAGACTACTGTA PO miR-199-as 343118 1904 TCATACAGCTAGATAACCAAAGA PO miR-9 343119 1773 ACAAGTGCCTTCACTGCAGT PO miR-17 343120 1871 GCATTATTACTCACGGTACGA PO miR-126a 343121 1787 ACCTAATATATCAAACATATCA PO miR-190 343122 1766 AAGCCCAAAAGGAGAATTCTTTG PO miR-186 343123 1839 CCTATCTCCCCTCTGGACC PO miR-198a 343124 1806 AGCTGCTTTTGGGATTCCGTTG PO miR-191c 343125 760 CCACACACTTCCTTACATTCCA PO miR-206d 343126 761 ATCTGCACTGTCAGCACTTT PO miR-94 343127 762 ACCCTTATCAGTTCTCCGTCCA PO miR-184 343128 763 GCCAATATTTCTGTGCTGCTA PO miR-195 343129 764 CTGGGACTTTGTAGGCCAGTT PO miR-193 343130 1861 GAACTGCCTTTCTCTCCA PO miR-185 343131 1786 ACCCTCCACCATGCAAGGGATG PO miR-188 343132 1879 GCTGGGTGGAGAAGGTGGTGAA PO miR-197a 343133 1906 TCCACATGGAGTTGCTGTTACA PO miR-194 343134 1771 ACAAGCTTTTTGCTCGTCTTAT PO miR-208 343135 938 AGACACGTGCACTGTAGA PO miR-139 343136 1887 GTCATCATTACCAGGCAGTATTA PO miR-200b 343137 1831 CATCGTTACCAGACAGTGTTA PO miR-200a 343138 291 CTACCATAGGGTAAAACCACT PS mir-140 343139 292 GCTGCAAACATCCGACTGAAAG PS mir-30a 343140 293 ACAACCAGCTAAGACACTGCCA PS mir-34 343141 294 AACACTGATTTCAAATGGTGCTA PS mir-29b 343142 295 CGCCAATATTTACGTGCTGCTA PS mir-16 343143 296 CTAGTGGTCCTAAACATTTCAC PS mir-203 343144 297 AACAAAATCACTAGTCTTCCA PS mir-7 343145 298 ACAAATTCGGTTCTACAGGGTA PS mir-10b 343146 299 AAAAGAGACCGGTTCACTGTGA PS mir-128a 343147 300 TCACTTTTGTGACTATGCAA PS mir-153 343148 301 CAGAACTTAGCCACTGTGAA PS mir-27b 343149 302 GCAAAAATGTGCTAGTGCCAAA PS mir-96 343150 303 ACTACCTGCACTGTAAGCACTTTG PS mir-17as/mir-91 343151 304 CGCGTACCAAAAGTAATAATG PS mir-123/mir-126as 343152 305 GCGACCATGGCTGTAGACTGTTA PS mir-132 343153 306 AATGCCCCTAAAAATCCTTAT PS mir-108 343154 307 GTGGTAATCCCTGGCAATGTGAT PS mir-23b 343155 308 AGCACAAACTACTACCTCA PS let-7i 343156 309 GGCCGTGACTGGAGACTGTTA PS mir-212 343157 310 ACTTTCGGTTATCTAGCTTTA PS mir-131 343158 311 AACCACACAACCTACTACCTCA PS let-7b 343159 312 ATACATACTTCTTTACATTCCA PS mir-1d 343160 313 ACAAACACCATTGTCACACTCCA PS mir-122a 343161 314 ACAGTTCTTCAACTGGCAGCTT PS mir-22 343162 315 ACAGGCCGGGACAAGTGCAATA PS mir-92 343163 316 GTAGTGCTTTCTACTTTATG PS mir-142 343164 317 CAGTGAATTCTACCAGTGCCATA PS mir-183 343165 318 CTGCCTGTCTGTGCCTGCTGT PS mir-214 343166 319 TGAGCTACAGTGCTTCATCTCA PS mir-143 343167 320 GGCTGTCAATTCATAGGTCAG PS mir-192 343168 321 AACTATACAACCTACTACCTCA PS let-7a 343169 322 ACTCACCGACAGCGTTGAATGTT PS mir-181a 343170 323 CAGACTCCGGTGGAATGAAGGA PS mir-205 343171 324 TCATAGCCCTGTACAATGCTGCT PS mir-103 343172 325 AGCCTATCCTGGATTACTTGAA PS mir-26a 343173 326 CAATGCAACTACAATGCAC PS mir-33a 343174 327 CCCAACAACATGAAACTACCTA PS mir-196 343175 328 TGATAGCCCTGTACAATGCTGCT PS mir-107 343176 329 GCTACCTGCACTGTAAGCACTTTT PS mir-106 343177 330 AACTATACAATCTACTACCTCA PS let-7f 343178 331 AACCGATTTCAAATGGTGCTAG PS mir-29c 343179 332 GCCCTTTTAACATTGCACTG PS mir-130a 343180 333 ACATGGTTAGATCAAGCACAA PS mir-218 343181 334 TGGCATTCACCGCGTGCCTTAA PS mir-124a 343182 335 TCAACATCAGTCTGATAAGCTA PS mir-21 343183 336 CTAGTACATCATCTATACTGTA PS mir-144 343184 337 GAAACCCAGCAGACAATGTAGCT PS mir-221 343185 338 GAGACCCAGTAGCCAGATGTAGCT PS mir-222 343186 339 CTTCCAGTCGGGGATGTTTACA PS mir-30d 343187 340 TCAGTTTTGCATGGATTTGCACA PS mir-19b 343188 341 GAAAGAGACCGGTTCACTGTGA PS mir-128b 343189 342 GCAAGCCCAGACCGCAAAAAG PS mir-129 343190 343 TAGCTGGTTGAAGGGGACCAA PS mir-133b 343191 344 ACTATGCAACCTACTACCTCT PS let-7d 343192 345 TGTAAACCATGATGTGCTGCTA PS mir-15b 343193 346 AACCGATTTCAGATGGTGCTAG PS mir-29a 343194 347 GAACAGATAGTCTAAACACTGGG PS mir-199b 343195 348 ACTATACAACCTCCTACCTCA PS let-7e 343196 349 AACCATACAACCTACTACCTCA PS let-7c 343197 350 AGGCATAGGATGACAAAGGGAA PS mir-204 343198 351 AAGGGATTCCTGGGAAAACTGGAC PS mir-145 343199 352 GGTACAATCAACGGTCGATGGT PS mir-213 343200 353 CTACCTGCACTATAAGCACTTTA PS mir-20 343201 354 ACAGCTGGTTGAAGGGGACCAA PS mir-133a 343202 355 GATTCACAACACCAGCT PS mir-138 343203 356 AACAATACAACTTACTACCTCA PS mir-98 343204 357 TCACAAGTTAGGGTCTCAGGGA PS mir-125b 343205 358 GAACAGGTAGTCTGAACACTGGG PS mir-199a 343206 359 AACCCACCGACAGCAATGAATGTT PS mir-181b 343207 360 CCATCTTTACCAGACAGTGTT PS mir-141 343208 361 TATCTGCACTAGATGCACCTTA PS mir-18 343209 362 AAAGTGTCAGATACGGTGTGG PS mir-220 343210 363 CTGTTCCTGCTGAACTGAGCCA PS mir-24 343211 364 AGGCGAAGGATGACAAAGGGAA PS mir-211 343212 365 TCAGTTATCACAGTACTGTA PS mir-101 343213 366 GCTGAGTGTAGGATGTTTACA PS mir-30b 343214 367 CACAAATTCGGATCTACAGGGTA PS mir-10a 343215 368 TCAGTTTTGCATAGATTTGCACA PS mir-19a 343216 369 CACAAACCATTATGTGCTGCTA PS mir-15a 343217 370 CTACGCGTATTCTTAAGCAATA PS mir-137 343218 371 AGAATTGCGTTTGGACAATCA PS mir-219 343219 372 ACAAAGTTCTGTGATGCACTGA PS mir-148b 343220 373 GCCCTTTCATCATTGCACTG PS mir-130b 343221 374 CACAGTTGCCAGCTGAGATTA PS mir-216 343222 375 CACAAGTTCGGATCTACGGGTT PS mir-100 343223 376 CCGGCTGCAACACAAGACACGA PS mir-187 343224 377 CAGCCGCTGTCACACGCACAG PS mir-210 343225 378 GTCTGTCAATTCATAGGTCAT PS mir-215 343226 379 GGGGTATTTGACAAACTGACA PS mir-223 343227 380 GCTGAGAGTGTAGGATGTTTACA PS mir-30c 343228 381 AACCTATCCTGAATTACTTGAA PS mir-26b 343229 382 CCAAGTTCTGTCATGCACTGA PS mir-152 343230 383 ATCACATAGGAATAAAAAGCCATA PS mir-135 343231 384 ATCCAATCAGTTCCTGATGCAGTA PS mir-217 343232 385 ACTGTACAAACTACTACCTCA PS let-7g 343233 386 CAATGCAACAGCAATGCAC PS mir-33b 343234 387 TGTGAGTTCTACCATTGCCAAA PS mir-182 343235 388 ACAAAGTTCTGTAGTGCACTGA PS mir-148a 343236 389 GGAAATCCCTGGCAATGTGAT PS mir-23a 343237 390 ACTCACCGACAGGTTGAATGTT PS mir-181c 343238 391 ACTGTAGGAATATGTTTGATA PS hypothetical miRNA-013 343239 392 ATTAAAAAGTCCTCTTGCCCA PS hypothetical miRNA-023 343240 393 GCTGCCGTATATGTGATGTCA PS hypothetical miRNA-030 343241 394 GGTAGGTGGAATACTATAACA PS hypothetical miRNA-033 343242 395 TAAACATCACTGCAAGTCTTA PS hypothetical miRNA-039 343243 396 TTGTAAGCAGTTTTGTTGACA PS hypothetical miRNA-040 343244 397 TCACAGAGAAAACAACTGGTA PS hypothetical miRNA-041 343245 398 CCTCTCAAAGATTTCCTGTCA PS hypothetical miRNA-043 343246 399 TGTCAGATAAACAGAGTGGAA PS hypothetical miRNA-044 343247 400 GAGAATCAATAGGGCATGCAA PS hypothetical miRNA-055 343248 401 AAGAACATTAAGCATCTGACA PS hypothetical miRNA-058 343249 402 AATCTCTGCAGGCAAATGTGA PS hypothetical miRNA-070 343250 403 AAACCCCTATCACGATTAGCA PS hypothetical miRNA-071 343251 404 GCCCCATTAATATTTTAACCA PS hypothetical miRNA-075 343252 405 CCCAATATCAAACATATCA PS hypothetical miRNA-079 343253 406 TATGATAGCTTCCCCATGTAA PS hypothetical miRNA-083 343254 407 CCTCAATTATTGGAAATCACA PS hypothetical miRNA-088 343255 408 ATTGATGCGCCATTTGGCCTA PS hypothetical miRNA-090 343256 409 CTGTGACTTCTCTATCTGCCT PS hypothetical miRNA-099 343257 410 AAACTTGTTAATTGACTGTCA PS hypothetical miRNA-101 343258 411 AAAGAAGTATATGCATAGGAA PS hypothetical miRNA-105 343259 412 GATAAAGCCAATAAACTGTCA PS hypothetical miRNA-107 343260 413 TCCGAGTCGGAGGAGGAGGAA PS hypothetical miRNA-111 343261 414 ATCATTACTGGATTGCTGTAA PS hypothetical miRNA-120 343262 415 CAAAAATTATCAGCCAGTTTA PS hypothetical miRNA-137 343263 416 AATCTCATTTTCATACTTGCA PS hypothetical miRNA-138 343264 417 AGAAGGTGGGGAGCAGCGTCA PS hypothetical miRNA-142 343265 418 CAAAATTGCAAGCAAATTGCA PS hypothetical miRNA-143 343266 419 TCCACAAAGCTGAACATGTCT PS hypothetical miRNA-144 343267 420 TATTATCAGCATCTGCTTGCA PS hypothetical miRNA-153 343268 421 AATAACACACATCCACTTTAA PS hypothetical miRNA-154 343269 422 AAGAAGGAAGGAGGGAAAGCA PS hypothetical miRNA-156 343270 423 ATGACTACAAGTTTATGGCCA PS hypothetical miRNA-161 343271 424 CAAAACATAAAAATCCTTGCA PS hypothetical miRNA-164 343272 425 TTACAGGTGCTGCAACTGGAA PS hypothetical miRNA-166 343273 426 AGCAGGTGAAGGCACCTGGCT PS hypothetical miRNA-168 343274 427 TATGAAATGCCAGAGCTGCCA PS hypothetical miRNA-169 343275 428 CCAAGTGTTAGAGCAAGATCA PS hypothetical miRNA-170 343276 429 AACGATAAAACATACTTGTCA PS hypothetical miRNA-171 343277 430 AGTAACTTCTTGCAGTTGGA PS hypothetical miRNA-172 343278 431 AGCCTCCTTCTTCTCGTACTA PS hypothetical miRNA-173 343279 432 ACCTCAGGTGGTTGAAGGAGA PS hypothetical miRNA-175 343280 433 ATATGTCATATCAAACTCCTA PS hypothetical miRNA-176 343281 434 GTGAGAGTAGCATGTTTGTCT PS hypothetical miRNA-177 343282 435 TGAAGGTTCGGAGATAGGCTA PS hypothetical miRNA-178 343283 436 AATTGGACAAAGTGCCTTTCA PS hypothetical miRNA-179 343284 437 ACCGAACAAAGTCTGACAGGA PS hypothetical miRNA-180 343285 438 AACTACTTCCAGAGCAGGTGA PS hypothetical miRNA-181 343286 439 GTAAGCGCAGCTCCACAGGCT PS hypothetical miRNA-183 343287 440 GAGCTGCTCAGCTGGCCATCA PS hypothetical miRNA-185 343288 441 TACTTTTCATTCCCCTCACCA PS hypothetical miRNA-188 343289 236 TAGCTTATCAGACTGATGTTGA PS miR-104 (Mourelatos) 343290 1780 ACAGGAGTCTGAGCATTTGA PS miR-105 (Mourelatos) 343291 1882 GGAACTTAGCCACTGTGAA PS miR-27 (Mourelatos) 343292 848 CTACCTGCACGAACAGCACTTT PS miR-93 (Mourelatos) 343293 855 TGCTCAATAAATACCCGTTGAA PS miR-95 (Mourelatos) 343294 1821 CACAAGATCGGATCTACGGGTT PS miR-99 (Mourelatos) 343295 1903 TCAGACCGAGACAAGTGCAATG PS miR-25 (Tuschl) 343296 1853 CTCAATAGACTGTGAGCTCCTT PS miR-28 (Tuschl) 343297 1825 CAGCTATGCCAGCATCTTGCC PS miR-31 (Tuschl) 343298 1865 GCAACTTAGTAATGTGCAATA PS miR-32 (Tuschl) 343299 854 GGAGTGAAGACACGGAGCCAGA PS miR-149 343300 1845 CGCAAGGTCGGTTCTACGGGTG PS miR-99b 343301 852 CACAGGTTAAAGGGTCTCAGGGA PS miR-125a 343302 853 AGCCAAGCTCAGACGGATCCGA PS miR-127 343303 1909 TCCATCATCAAAACAAATGGAGT PS miR-136 343304 1843 CGAAGGCAACACGGATAACCTA PS miR-154 343305 1880 GCTTCCAGTCGAGGATGTTTACA PS miR-30a-s 343306 1911 TCCGTGGTTCTACCCTGTGGTA PS miR-140-as 343307 1836 CCATAAAGTAGGAAACACTACA PS miR-142-as 343308 1761 AACAGGTAGTCTGAACACTGGG PS miR-199-s 343309 1762 AACCAATGTGCAGACTACTGTA PS miR-199-as 343310 1904 TCATACAGCTAGATAACCAAAGA PS miR-9 343311 1773 ACAAGTGCCTTCACTGCAGT PS miR-17 343312 1871 GCATTATTACTCACGGTACGA PS miR-126a 343313 1787 ACCTAATATATCAAACATATCA PS miR-190 343314 1766 AAGCCCAAAAGGAGAATTCTTTG PS miR-186 343315 1839 CCTATCTCCCCTCTGGACC PS miR-198a 343316 1806 AGCTGCTTTTGGGATTCCGTTG PS miR-191c 343317 760 CCACACACTTCCTTACATTCCA PS miR-206d 343318 761 ATCTGCACTGTCAGCACTTT PS miR-94 343319 762 ACCCTTATCAGTTCTCCGTCCA PS miR-184 343320 763 GCCAATATTTCTGTGCTGCTA PS miR-195 343321 764 CTGGGACTTTGTAGGCCAGTT PS miR-193 343322 1861 GAACTGCCTTTCTCTCCA PS miR-185 343323 1786 ACCCTCCACCATGCAAGGGATG PS miR-188 343324 1879 GCTGGGTGGAGAAGGTGGTGAA PS miR-197a 343325 1906 TCCACATGGAGTTGCTGTTACA PS miR-194 343326 1771 ACAAGCTTTTTGCTCGTCTTAT PS miR-208 343327 938 AGACACGTGCACTGTAGA PS miR-139 343328 1887 GTCATCATTACCAGGCAGTATTA PS miR-200b 343329 1831 CATCGTTACCAGACAGTGTTA PS miR-200a 344290 1774 ACACAAATTCGGTTCTACAGGG PO miR-10b (Tuschl) 344292 1867 GCACGAACAGCACTTTG PO miR-93 (Tuschl) 344293 1770 ACAAGATCGGATCTACGGGT PO miR-99a (Tuschl) 344297 1912 TCTAGTGGTCCTAAACATTTCA PO miR-203 (Tuschl) 344298 1828 CAGGCATAGGATGACAAAGGGAA PO miR-204 (Tuschl) 344299 1767 AATACATACTTCTTTACATTCCA PO miR-1d (Tuschl) 344300 1769 ACAAATTCGGATCTACAGGGTA PS miR-10 (Tuschl) 344301 1774 ACACAAATTCGGTTCTACAGGG PS miR-10b (Tuschl) 344302 1890 TAACCGATTTCAAATGGTGCTA PS miR-29c (Tuschl) 344303 1867 GCACGAACAGCACTTTG PS miR-93 (Tuschl) 344304 1770 ACAAGATCGGATCTACGGGT PS miR-99a (Tuschl) 344305 1816 CAAACACCATTGTCACACTCCA PS miR-122a, b (Tuschl) 344306 1920 TGTCAATTCATAGGTCAG PS miR-192 (Tuschl) 344307 1832 CCAACAACATGAAACTACCTA PS miR-196 (Tuschl) 344308 1912 TCTAGTGGTCCTAAACATTTCA PS miR-203 (Tuschl) 344309 1828 CAGGCATAGGATGACAAAGGGAA PS miR-204 (Tuschl) 344310 1767 AATACATACTTCTTTACATTCCA PS miR-1d (Tuschl) 344354 1812 ATGCCCTTTTAACATTGCACTG PO mir-130 (Kosik) 344356 1921 TGTCCGTGGTTCTACCCTGTGGTA PO mir-239* (Kosik) 344358 1814 ATGCTTTTTGGGGTAAGGGCTT PO mir-129as/mir-258* (Kosik) 344359 1811 ATGCCCTTTCATCATTGCACTG PO mir-266* (Kosik) 344360 1918 TGGCATTCACCGCGTGCCTTA PS mir-124a (Kosik) 344361 1754 AAAGAGACCGGTTCACTGTGA PS mir-128 (Kosik) 344362 1812 ATGCCCTTTTAACATTGCACTG PS mir-130 (Kosik) 344363 1854 CTCACCGACAGCGTTGAATGTT PS mir-178 (Kosik) 344364 1921 TGTCCGTGGTTCTACCCTGTGGTA PS mir-239* (Kosik) 344365 1823 CACATGGTTAGATCAAGCACAA PS mir-253* (Kosik) 344366 1814 ATGCTTTTTGGGGTAAGGGCTT PS mir-129as/mir-258* (Kosik) 344367 1811 ATGCCCTTTCATCATTGCACTG PS mir-266* (Kosik) 344625 1785 ACATTTTTCGTTATTGCTCTTGA PO mir-240* (Kosik) 344626 1790 ACGGAAGGGCAGAGAGGGCCAG PO mir-232* (Kosik) 344627 1775 ACACCAATGCCCTAGGGGATGCG PO mir-227* (Kosik) 344628 1834 CCAGCAGCACCTGGGGCAGT PO mir-226* (Kosik) 344629 1900 TCAACAAAATCACTGATGCTGGA PO mir-244* (Kosik) 344630 1800 AGAGGTCGACCGTGTAATGTGC PO mir-224* (Kosik) 344631 1862 GACGGGTGCGATTTCTGTGTGAGA PO mir-248* (Kosik) 344632 1785 ACATTTTTCGTTATTGCTCTTGA PS mir-240* (Kosik) 344633 1790 ACGGAAGGGCAGAGAGGGCCAG PS mir-232* (Kosik) 344634 1775 ACACCAATGCCCTAGGGGATGCG PS mir-227* (Kosik) 344635 1834 CCAGCAGCACCTGGGGCAGT PS mir-226* (Kosik) 344636 1900 TCAACAAAATCACTGATGCTGGA PS mir-244* (Kosik) 344637 1800 AGAGGTCGACCGTGTAATGTGC PS mir-224* (Kosik) 344638 1862 GACGGGTGCGATTTCTGTGTGAGA PS mir-248* (Kosik) 345527 1827 CAGCTTTCAAAATGATCTCAC PO miR-Bantam 345529 1897 TAGGAGAGAGAAAAAGACTGA PS miR-14 345531 1827 CAGCTTTCAAAATGATCTCAC PS miR-Bantam 345708 1897 TAGGAGAGAGAAAAAGACTGA PO miR-14 346721 1884 GGCGGAACTTAGCCACTGTGAA PO miR-27a (RFAM-Human) 346722 1857 CTTCAGTTATCACAGTACTGTA PO miR-101 (RFAM-Human) 346727 1802 AGCAAGCCCAGACCGCAAAAAG PO miR-129b (RFAM-Human) 346728 1898 TAGTTGGCAAGTCTAGAACCA PO miR-182* (RFAM-Human) 346729 1830 CATCATTACCAGGCAGTATTAGAG PO miR-200a (RFAM-Human) 346730 1792 ACTGATATCAGCTCAGTAGGCAC PO miR-189 (RFAM-Human) 346731 1870 GCAGAAGCATTTCCACACAC PO miR-147 (RFAM-Human) 346732 1889 TAAACGGAACCACTAGTGACTTG PO miR-224 (RFAM-Human) 346733 1838 CCCTCTGGTCAACCAGTCACA PO miR-134 (RFAM-Human) 346734 1763 AACCCATGGAATTCAGTTCTCA PO miR-146 (RFAM-Human) 346735 1824 CACTGGTACAAGGGTTGGGAGA PO miR-150 (RFAM-Human) 346736 1893 TACCTGCACTATAAGCACTTTA PS mir-20 346737 1788 ACCTATCCTGAATTACTTGAA PS mir-26b 346738 1884 GGCGGAACTTAGCCACTGTGAA PS miR-27a (RFAM-Human) 346739 1857 CTTCAGTTATCACAGTACTGTA PS miR-101 (RFAM-Human) 346740 1793 ACTGATTTCAAATGGTGCTA PS mir-29b 346741 1847 CGGCTGCAACACAAGACACGA PS miR-187 (RFAM-Human) 346742 1844 CGACCATGGCTGTAGACTGTTA PS miR-132 (RFAM-Human) 346743 1901 TCACATAGGAATAAAAAGCCATA PS miR-135 (RFAM-Human) 346744 1802 AGCAAGCCCAGACCGCAAAAAG PS miR-129b (RFAM-Human) 346745 1898 TAGTTGGCAAGTCTAGAACCA PS miR-182* (RFAM-Human) 346746 1830 CATCATTACCAGGCAGTATTAGAG PS miR-200a (RFAM-Human) 346747 1792 ACTGATATCAGCTCAGTAGGCAC PS miR-189 (RFAM-Human) 346748 1870 GCAGAAGCATTTCCACACAC PS miR-147 (RFAM-Human) 346749 1889 TAAACGGAACCACTAGTGACTTG PS miR-224 (RFAM-Human) 346750 1838 CCCTCTGGTCAACCAGTCACA PS miR-134 (RFAM-Human) 346751 1763 AACCCATGGAATTCAGTTCTCA PS miR-146 (RFAM-Human) 346752 1824 CACTGGTACAAGGGTTGGGAGA PS miR-150 (RFAM-Human) 346939 1907 TCCAGTCAAGGATGTTTACA PO miR-30e (RFAM-M. musculus) 346940 1781 ACAGGATTGAGGGGGGGCCCT PO miR-296 (RFAM-M. musculus) 346941 1815 ATGTATGTGGGACGGTAAACCA PO miR-299 (RFAM-M. musculus) 346942 1881 GCTTTGACAATACTATTGCACTG PO miR-301 (RFAM-M. musculus) 346943 1902 TCACCAAAACATGGAAGCACTTA PO miR-302 (RFAM-M. musculus) 346944 1866 GCAATCAGCTAACTACACTGCCT PO miR-34a (RFAM-M. musculus) 346945 1776 ACACTGATTTCAAATGGTGCTA PO miR-29b (RFAM-M. musculus) 346947 1795 AGAAAGGCAGCAGGTCGTATAG PO let-7d* (RFAM-M. musculus) 346948 1810 ATCTGCACTGTCAGCACTTTA PO miR-106b (RFAM-M. musculus) 346949 1784 ACATCGTTACCAGACAGTGTTA PO miR-200a (RFAM-M. musculus) 346950 1874 GCGGAACTTAGCCACTGTGAA PO miR-27a (RFAM-M. musculus) 346951 1826 CAGCTATGCCAGCATCTTGCCT PO miR-31 (RFAM-M. musculus) 346954 1801 AGCAAAAATGTGCTAGTGCCAAA PO miR-96 (RFAM-M. musculus) 346955 1759 AACAACCAGCTAAGACACTGCCA PO miR-172 (RFAM-M. musculus) 346956 1907 TCCAGTCAAGGATGTTTACA PS miR-30e (RFAM-M. musculus) 346957 1781 ACAGGATTGAGGGGGGGCCCT PS miR-296 (RFAM-M. musculus) 346958 1815 ATGTATGTGGGACGGTAAACCA PS miR-299 (RFAM-M. musculus) 346959 1881 GCTTTGACAATACTATTGCACTG PS miR-301 (RFAM-M. musculus) 346960 1902 TCACCAAAACATGGAAGCACTTA PS miR-302 (RFAM-M. musculus) 346961 1866 GCAATCAGCTAACTACACTGCCT PS miR-34a (RFAM-M. musculus) 346962 1776 ACACTGATTTCAAATGGTGCTA PS miR-29b (RFAM-M. musculus) 346963 1851 CTAGTGGTCCTAAACATTTCA PS miR-203 (RFAM-M. musculus) 346964 1795 AGAAAGGCAGCAGGTCGTATAG PS let-7d* (RFAM-M. musculus) 346965 1810 ATCTGCACTGTCAGCACTTTA PS miR-106b (RFAM-M. musculus) 346966 1784 ACATCGTTACCAGACAGTGTTA PS miR-200a (RFAM-M. musculus) 346967 1874 GCGGAACTTAGCCACTGTGAA PS miR-27a (RFAM-M. musculus) 346968 1826 CAGCTATGCCAGCATCTTGCCT PS miR-31 (RFAM-M. musculus) 346969 1829 CAGGCCGGGACAAGTGCAATA PS miR-92 (RFAM-M. musculus) 346970 1849 CTACCTGCACGAACAGCACTTTG PS miR-93 (RFAM-M. musculus) 346971 1801 AGCAAAAATGTGCTAGTGCCAAA PS miR-96 (RFAM-M. musculus) 346972 1759 AACAACCAGCTAAGACACTGCCA PS miR-172 (RFAM-M. musculus) 348169 1922 TTCGCCCTCTCAACCCAGCTTTT PO miR-320 348170 1860 GAACCCACAATCCCTGGCTTA PO miR-321-1 348172 1908 TCCATAAAGTAGGAAACACTACA PO miR-142as (Michael et al) 348175 1905 TCATCATTACCAGGCAGTATTA PO miR-200b (Michael et al) 348177 1820 CACAAATTCGGTTCTACAGGGTA PO miR-10b (Michael et al) 348178 1878 GCTGGATGCAAACCTGCAAAACT PO miR-19b (Michael et al) 348180 1869 GCAGAACTTAGCCACTGTGAA PO miR-27* (Michael et al) 348181 1858 CTTCCAGTCAAGGATGTTTACA PO miR-97 (Michael et al) 348182 1855 CTGGCTGTCAATTCATAGGTCA PO miR-192 (Michael et al) 348183 1922 TTCGCCCTCTCAACCCAGCTTTT PS miR-320 348184 1860 GAACCCACAATCCCTGGCTTA PS miR-321-1 348185 1886 GTAAACCATGATGTGCTGCTA PS miR-15b (Michael et al) 348186 1908 TCCATAAAGTAGGAAACACTACA PS miR-142as (Michael et al) 348188 1883 GGATTCCTGGGAAAACTGGAC PS miR-145 (Michael et al) 348189 1905 TCATCATTACCAGGCAGTATTA PS miR-200b (Michael et al) 348190 1791 ACTATACAATCTACTACCTCA PS let-7f (Michael et al) 348191 1820 CACAAATTCGGTTCTACAGGGTA PS miR-10b (Michael et al) 348192 1878 GCTGGATGCAAACCTGCAAAACT PS miR-19b (Michael et al) 348193 1873 GCCTATCCTGGATTACTTGAA PS miR-26a (Michael et al) 348194 1869 GCAGAACTTAGCCACTGTGAA PS miR-27* (Michael et al) 348195 1858 CTTCCAGTCAAGGATGTTTACA PS miR-97 (Michael et al) 348196 1855 CTGGCTGTCAATTCATAGGTCA PS miR-192 (Michael et al) 354168 1751 AAACCACACAACCTACTACCTCA PS let-7b-Ruvkun 354169 1752 AAACCATACAACCTACTACCTCA PS let-7c-Ruvkun 354170 1764 AACTATGCAACCTACTACCTCT PS let-7d-Ruvkun 354171 1765 AACTGTACAAACTACTACCTCA PS let-7gL-Ruvkun 354172 1760 AACAGCACAAACTACTACCTCA PS let-7i-Ruvkun 354173 1924 TTGGCATTCACCGCGTGCCTTAA PS mir-124a-Ruvkun 354174 1833 CCAAGCTCAGACGGATCCGA PS mir-127-Ruvkun 354175 1896 TACTTTCGGTTATCTAGCTTTA PS mir-131-Ruvkun 354176 1846 CGGCCTGATTCACAACACCAGCT PS mir-138-Ruvkun 354177 1768 ACAAACCATTATGTGCTGCTA PS mir-15-Ruvkun 354178 1789 ACGCCAATATTTACGTGCTGCTA PS mir-16-Ruvkun 354179 1852 CTATCTGCACTAGATGCACCTTA PS mir-18-Ruvkun 354180 1779 ACAGCTGCTTTTGGGATTCCGTTG PS mir-191-Ruvkun 354181 1891 TAACCGATTTCAGATGGTGCTA PS mir-29a-Ruvkun 354182 1813 ATGCTTTGACAATACTATTGCACTG PS mir-301-Ruvkun 354183 1805 AGCTGAGTGTAGGATGTTTACA PS mir-30b-Ruvkun 354184 1804 AGCTGAGAGTGTAGGATGTTTACA PS mir-30c-Ruvkun 354185 1807 AGCTTCCAGTCGGGGATGTTTACA PS mir-30d-Ruvkun 354186 1835 CCAGCAGCACCTGGGGCAGTGG PS mir-324-3p-Ruvkun 354187 1899 TATGGCAGACTGTGATTTGTTG PS mir-7-1*-Ruvkun 354188 1850 CTACCTGCACTGTAAGCACTTTG PS mir-91-Ruvkun 354189 1822 CACATAGGAATGAAAAGCCATA PS mir-135b (Ruvkun) 354190 1895 TACTAGACTGTGAGCTCCTCGA PS mir-151* (Ruvkun) 354191 1885 GGCTATAAAGTAACTGAGACGGA PS mir-340 (Ruvkun) 354192 1923 TTCTAGGATAGGCCCAGGGGC PS mir-331 (Ruvkun) 354193 1892 TACATACTTCTTTACATTCCA PS miR-1 (RFAM) 354194 1817 CAATCAGCTAACTACACTGCCT PS miR-34c (RFAM) 354195 1837 CCCCTATCACGATTAGCATTAA PS miR-155 (RFAM) 354196 1910 TCCATCATTACCCGGCAGTATT PS miR-200c (RFAM) 354197 1818 CAATCAGCTAATGACACTGCCT PS miR-34b (RFAM) 354198 1753 AAACCCAGCAGACAATGTAGCT PS mir-221 (RFAM-M. musculus) 354199 1796 AGACCCAGTAGCCAGATGTAGCT PS mir-222 (RFAM-M. musculus) 354200 1917 TGAGCTCCTGGAGGACAGGGA PS mir-339-1 (RFAM) 354201 1925 TTTAAGTGCTCATAATGCAGT PS miR-20* (human) 354202 1926 TTTTCCCATGCCCTATACCTCT PS miR-202 (human) 354203 1856 CTTCAGCTATCACAGTACTGTA PS miR-101b 354204 1894 TACCTGCACTGTTAGCACTTTG PS miR-106a 354205 1772 ACAAGTGCCCTCACTGCAGT PS miR-17-3p 354206 1859 GAACAGGTAGTCTAAACACTGGG PS miR-199b (mouse) 354207 1915 TCTTCCCATGCGCTATACCTCT PS miR-202 (mouse) 354208 1808 AGGCAAAGGATGACAAAGGGAA PS miR-211 (mouse) 354209 1809 ATCCAGTCAGTTCCTGATGCAGTA PS miR-217 (mouse) 354210 1888 TAAACGGAACCACTAGTGACTTA PS miR-224 (RFAM-mouse) 354211 1758 AACAAAATCACAAGTCTTCCA PS miR-7b 354212 1919 TGTAAGTGCTCGTAATGCAGT PS miR-20* (mouse) 354213 1778 ACACTTACTGGACACCTACTAGG PS mir-325 (human) 354214 1777 ACACTTACTGAGCACCTACTAGG PS mir-325 (mouse) 354215 1877 GCTGGAGGAAGGGCCCAGAGG PS mir-326 (human) 354216 1794 ACTGGAGGAAGGGCCCAGAGG PS mir-326 (mouse) 354217 1755 AAAGAGGTTAACCAGGTGTGTT PS mir-329-1 (human) 354218 1750 AAAAAGGTTAGCTGGGTGTGTT PS mir-329-1 (mouse) 354219 1914 TCTCTGCAGGCCGTGTGCTTTGC PS mir-330 (human) 354220 1913 TCTCTGCAGGCCCTGTGCTTTGC PS mir-330 (mouse) 354221 1757 AAAGGCATCATATAGGAGCTGGA PS mir-337 (human) 354222 1756 AAAGGCATCATATAGGAGCTGAA PS mir-337 (mouse) 354223 1872 GCCCTGGACTAGGAGTCAGCA PS mir-345 (human) 354224 1868 GCACTGGACTAGGGGTCAGCA PS mir-345 (mouse) 354225 1799 AGAGGCAGGCATGCGGGCAGACA PS mir-346 (human) 354226 1798 AGAGGCAGGCACTCGGGCAGACA PS mir-346 (mouse) 354228 1841 CCTCAAGGAGCCTCAGTCTAGT PS miR-151 (rat) 354229 1797 AGAGGCAGGCACTCAGGCAGACA PS miR-346 (rat) 354230 1819 CAATCAGCTAATTACACTGCCTA PS miR-34b (mouse) 354231 1842 CCTCAAGGAGCTTCAGTCTAGT PS miR-151 (human) 354232 1751 AAACCACACAACCTACTACCTCA PO let-7b-Ruvkun 354234 1764 AACTATGCAACCTACTACCTCT PO let-7d-Ruvkun 354235 1765 AACTGTACAAACTACTACCTCA PO let-7gL-Ruvkun 354236 1760 AACAGCACAAACTACTACCTCA PO let-7i-Ruvkun 354238 1833 CCAAGCTCAGACGGATCCGA PO mir-127-Ruvkun 354239 1896 TACTTTCGGTTATCTAGCTTTA PO mir-131-Ruvkun 354240 1846 CGGCCTGATTCACAACACCAGCT PO mir-138-Ruvkun 354242 1789 ACGCCAATATTTACGTGCTGCTA PO mir-16-Ruvkun 354243 1852 CTATCTGCACTAGATGCACCTTA PO mir-18-Ruvkun 354244 1779 ACAGCTGCTTTTGGGATTCCGTTG PO mir-191-Ruvkun 354245 1891 TAACCGATTTCAGATGGTGCTA PO mir-29a-Ruvkun 354246 1813 ATGCTTTGACAATACTATTGCACTG PO mir-301-Ruvkun 354248 1804 AGCTGAGAGTGTAGGATGTTTACA PO mir-30c-Ruvkun 354250 1835 CCAGCAGCACCTGGGGCAGTGG PO mir-324-3p-Ruvkun 354251 1899 TATGGCAGACTGTGATTTGTTG PO mir-7-1*-Ruvkun 354253 1822 CACATAGGAATGAAAAGCCATA PO mir-135b (Ruvkun) 354254 1895 TACTAGACTGTGAGCTCCTCGA PO mir-151* (Ruvkun) 354255 1885 GGCTATAAAGTAACTGAGACGGA PO mir-340 (Ruvkun) 354256 1923 TTCTAGGATAGGCCCAGGGGC PO mir-331 (Ruvkun) 354258 1817 CAATCAGCTAACTACACTGCCT PO miR-34c (RFAM) 354259 1837 CCCCTATCACGATTAGCATTAA PO miR-155 (RFAM) 354260 1910 TCCATCATTACCCGGCAGTATT PO miR-200c (RFAM) 354261 1818 CAATCAGCTAATGACACTGCCT PO miR-34b (RFAM) 354264 1917 TGAGCTCCTGGAGGACAGGGA PO mir-339-1 (RFAM) 354265 1925 TTTAAGTGCTCATAATGCAGT PO miR-20* (human) 354266 1926 TTTTCCCATGCCCTATACCTCT PO miR-202 (human) 354267 1856 CTTCAGCTATCACAGTACTGTA PO miR-101b 354268 1894 TACCTGCACTGTTAGCACTTTG PO miR-106a 354269 1772 ACAAGTGCCCTCACTGCAGT PO miR-17-3p 354270 1859 GAACAGGTAGTCTAAACACTGGG PO miR-199b (mouse) 354271 1915 TCTTCCCATGCGCTATACCTCT PO miR-202 (mouse) 354272 1808 AGGCAAAGGATGACAAAGGGAA PO miR-211 (mouse) 354273 1809 ATCCAGTCAGTTCCTGATGCAGTA PO miR-217 (mouse) 354274 1888 TAAACGGAACCACTAGTGACTTA PO miR-224 (RFAM-mouse) 354275 1758 AACAAAATCACAAGTCTTCCA PO miR-7b 354276 1919 TGTAAGTGCTCGTAATGCAGT PO miR-20* (mouse) 354277 1778 ACACTTACTGGACACCTACTAGG PO mir-325 (human) 354278 1777 ACACTTACTGAGCACCTACTAGG PO mir-325 (mouse) 354279 1877 GCTGGAGGAAGGGCCCAGAGG PO mir-326 (human) 354280 1794 ACTGGAGGAAGGGCCCAGAGG PO mir-326 (mouse) 354281 1755 AAAGAGGTTAACCAGGTGTGTT PO mir-329-1 (human) 354282 1750 AAAAAGGTTAGCTGGGTGTGTT PO mir-329-1 (mouse) 354283 1914 TCTCTGCAGGCCGTGTGCTTTGC PO mir-330 (human) 354284 1913 TCTCTGCAGGCCCTGTGCTTTGC PO mir-330 (mouse) 354285 1757 AAAGGCATCATATAGGAGCTGGA PO mir-337 (human) 354286 1756 AAAGGCATCATATAGGAGCTGAA PO mir-337 (mouse) 354287 1872 GCCCTGGACTAGGAGTCAGCA PO mir-345 (human) 354288 1868 GCACTGGACTAGGGGTCAGCA PO mir-345 (mouse) 354289 1799 AGAGGCAGGCATGCGGGCAGACA PO mir-346 (human) 354290 1798 AGAGGCAGGCACTCGGGCAGACA PO mir-346 (mouse) 354292 1841 CCTCAAGGAGCCTCAGTCTAGT PO miR-151 (rat) 354293 1797 AGAGGCAGGCACTCAGGCAGACA PO miR-346 (rat) 354294 1819 CAATCAGCTAATTACACTGCCTA PO miR-34b (mouse) 354295 1842 CCTCAAGGAGCTTCAGTCTAGT PO miR-151 (human)

It is also understood that, although many of the oligomeric compounds listed in Tables 64-66 have been designed to target or mimic a particular miRNA from humans, for example, that oligomeric compound may also target or mimic other miRNAs from mammals, such as those from rodent species, for example. It is also understood that these miRNAs and mimics can serve as the basis for several variations of nucleic acid oligomeric compounds, including compounds with chemical modifications such as uniform or chimeric 2′-MOE oligomeric compounds, as well as LNAs and PNAs; such oligomeric compounds are also within the scope of the invention. One such non-limiting example is ISIS Number 351104 (CTAGTGGTCCTAAACATTTCAC; SEQ ID NO: 296), which is a PNA oligomeric compound targeted to the human mir-203 miRNA.

Example 35 Targeting miRNAs in Introns and Exons

By mapping the coding sequences of miRNAs onto genomic contigs (which sequence information is available from public databases, such as GenBank and Locus Link), and identifying loci at which other reported gene coding sequences also co-map, it was observed that miRNAs can be encoded within the exons or introns of other genes. The oligomeric compounds of the present invention can be designed to target introns and exons of these genes. For example, the oligomeric compounds of the present invention can be designed to target introns or exons of the genes listed in Table 67. More specifically, these oligomeric compounds can target the miRNAs encoded within the exons or introns of these genes listed in Table 67.

TABLE 67 Oligomeric compounds targeting miRNAs found within introns or exons SEQ ID Locus SEQ ISIS # NO: Locus containing miRNA ID NO 327873 291 Ubiquitin protein ligase WWP2 containing mir- 1928 140 327874 292 hypothetical protein FLJ13189 1929 327877 295 deleted in lymphocytic leukemia, 2 containing 1930 mir-16-1 and mir-15a-1 327877 295 SMC4 (structural maintenance of chromosomes 4, 1931 yeast)-like 1 containing mir-16-3 and mir-15b 327879 297 heterogeneous nuclear ribonucleoprotein K 1932 containing mir-7-1 327879 297 pituitary gland specific factor 1a containing 1933 mir-7-3 327881 299 R3H domain (binds single-stranded nucleic 1934 acids) containing containing mir-128a 327882 300 protein tyrosine phosphatase, receptor type, N 1935 polypeptide 2 containing mir-153-2 327882 300 protein tyrosine phosphatase, receptor type, N 1936 containing mir-153-1 327883 301 chromosome 9 ORF3 containing mir-23b, mir-24-2 1937 and mir-27b 327892 310 Transcriptional activator of the c-fos 1938 promoter containing mir-131-1/miR-9 327896 314 hypothetical protein MGC14376 containing mir- 1939 22 327906 324 hypothetical protein FLJ11729 containing mir- 1940 103-2 327906 324 hypothetical protein FLJ12899 containing mir- 1941 103-1 327907 325 conserved gene amplified in osteosarcoma 1942 containing miR-26a-2 327907 325 HYA22 protein containing miR-26a-1 1943 327908 326 Sterol regulatory element binding 1944 transcription factor 2 containing mir-33a 327910 328 pantothenate kinase containing mir-107 1945 327912 330 upstream regulatory element binding protein 1 1946 containing mir-98 and let-7f-2 327915 333 slit (Drosophila) homolog 3 containing mir- 1947 218-2 327915 333 slit (Drosophila) homolog 2 containing mir- 1948 218-1 327923 341 cyclic AMP-regulated phosphoprotein, 21 kD 1949 containing mir-128b 327932 350 transient receptor potential cation channel, 1950 subfamily M, member 3 containing mir-204 327946 364 melastatin 1 containing mir-211 1951 327947 365 RNA cyclase homolog containing mir-101-3 1952 327954 372 CGI-120 protein containing mir-148b 1953 327963 381 nuclear LIM interactor-interacting factor 1954 containing mir-26b 327964 382 COPZ2 for nonclathrin coat protein zeta-COP 1955 containing mir-152 327967 385 hypothetical protein PRO2730 containing let-7g 1956 327968 386 sterol regulatory element-binding protein-1/ 1957 mir-33b 328089 391 talin 2 containing hypothetical miR-13/miR-190 1958 328091 393 calcitonin receptor containing hypothetical 1959 miRNA 30 328092 394 glutamate receptor, ionotrophic, AMPA 3/ 1960 hypothetical miRNA-033 328093 395 myosin, heavy polypeptide 7B, cardiac muscle, 1961 beta containing hypothetical miRNA 039 328101 403 LOC 114614/hypothetical miRNA-071 1962 328104 406 dachshund (Drosophila) homolog containing 1963 hypothetical miRNA 083 328105 407 DiGeorge syndrome critical region gene 8/ 1964 hypothetical miRNA-088 328111 413 hypothetical protein FLJ21016, containing 1965 hypothetical miRNA 111 328117 419 collagen, type I, alpha 1/hypothetical miRNA- 1966 144 328119 421 hypothetical protein HH114 containing 1967 hypothetical miRNA 154 328120 422 sprouty (Drosophila) homolog 4 containing 1968 hypothetical miRNA 156 328124 426 ribosomal protein L5/hypothetical miRNA 168-2 1969 328125 427 forkhead box P2/hypothetical miRNA 169 1970 328127 429 glutamate receptor, ionotropic, AMPA 2/ 1971 hypothetical miRNA 171 328128 430 potassium large conductance calcium-activated 1972 channel, subfamily M, alpha member 1 containing hypothetical miRNA 172 328131 433 hypothetical protein FLJ20307 1973 328135 437 cezanne 2/hypothetical miRNA-180 1974 328137 439 tight junction protein 1 (zona occludens 1)/ 1975 hypothetical miRNA-183 340343 1780 gamma-aminobutyric acid (GABA) A receptor, 1976 alpha 3 containing miR-105 (Mourelatos) and miR-105-2 340348 848 Minichromosome maintenance deficient (S. cerevisiae) 1977 7 containing miR-93 (Mourelatos) and miR-25 and miR-94 340350 855 KIAA1808 protein containing miR-95 1978 (Mourelatos) 340356 1853 LIM domain-containing preferred translocation 1979 partner in lipoma containing miR-28 340360 1865 chromosome 9 open reading frame 5 containing 1980 miR-32 341785 854 glypican 1 containing miR-149 1981 341798 1871 Notch 4 like containing mir-123/mir-126 1982 341800 1766 zinc finger protein 265 containing miR-186 1983 341801 1839 follistatin-like 1 containing miR-198 1984 341802 1806 hypothetical protein FLJ10496 containing miR- 1985 191 341808 1861 hypothetical protein DKFZp761P1121, containing 1986 miR-185 341809 1786 chloride channel 5 (nephrolithiasis 2, X- 1987 linked, Dent disease) containing miR-188 341812 1771 myosin, heavy polypeptide 6, cardiac muscle, 1988 alpha (cardiomyopathy, hypertrophic 1) containing miR-208 341813 938 phosphodiesterase 2A, cGMP-stimulated 1989 containing miR-139 344611 1785 mesoderm specific transcript (mouse) homolog 1990 containing mir-240* (Kosik) 344615 1900 Apoptosis-associated tyrosine kinase 1991 containing mir-244* (Kosik) 344617 1862 RNB6 containing mir-248* (Kosik) 1992 346692 1889 gamma-aminobutyric acid (GABA) A receptor, 1993 epsilon, containing miR-224 (Sanger) 348128 1858 Nuclear transcription factor Y, gamma 1994 containing miR-30c-2 and miR-30e

Example 36 Oligomeric Compounds Targeting Components of the RNAi Pathway

In one step of miRNA processing, the pre-miRNAs, approximately 70 to 110 nucleotides in length, are processed by the human Dicer RNase into mature miRNAs. The Dicer enzyme is conserved from fungi to vertebrates. The helicase-moi gene is the human homolog of Dicer from Drosophila. Human Dicer is required for the production of active small non-coding RNAs involved in repressing gene expression by the RNA interference pathway; targeted destruction in cultured human cells of the mRNA encoding human Dicer leads to accumulation of the let-7 pre-miRNA (Hutvagner, et al., 2001, Science 293(5531):834-8). Furthermore, the zebrafish Dicer1 ortholog was cloned and its expression disrupted by target-selected gene inactivation; in homozygous dicer1 mutants, an initial build-up of miRNA levels produced by maternal Dicer1 was observed, but miRNA accumulation halted after a few days, and a developmental arrest was observed at around day 10, indicating that miRNA-producing Dicer1 is essential for vertebrate development (Wienholds, et al., 2003, Nat. Genet., 35(3):217-8). The Dicer gene has also been disrupted in mice. Loss of Dicer1 led to lethality early in development, with Dicer1-null embryos depleted of stem cells. Coupled with the inability to generate viable Dicer1-null embryonic stem cells, this suggests a role for Dicer and, by implication, the RNAi machinery in maintaining the stem cell population during early mouse development (Bernstein, et al., 2003, Nat. Genet., 35(3):215-7).

Thus, it was predicted that treatment of cells with oligomeric compounds targeting human Dicer would result in an increase in expression levels of miRNA precursor structures, and thus would be useful in increasing the sensitivity of or enabling the detection of certain pre-miRNAs and/or pri-miRNAs otherwise beneath the limits of detection. It was also predicted that treatment of cells with oligomeric compounds targeting human Dicer would result in a decrease in mature miRNAs, leading to dysregulation of miRNA-regulated targets. Thus, a transcriptomics- or proteomics-based approach could be used to compare and identify target RNAs or proteins for which changes in expression levels correlate directly or inversely with the changes in mature miRNA levels. Target RNAs or their downstream protein products which are being misregulated upon treatment with oligomeric compounds targeting human Dicer, can thereby lead to the identification of any potential miRNA-regulated targets.

The present invention provides methods of maintaining a pluripotent stem cell comprising contacting the cell with an effective amount of an oligomeric compound targeting human Dicer. The pluripotent stem cell can be present in a sample of cord blood or bone marrow, or may be present as part of a cell line. In addition, the pluripotent stem cell can be an embryonic stem cell.

In some embodiments, oligomeric compounds ISIS Number 138648 (GCTGACCTTTTTGCTTCTCA; herein incorporated as SEQ ID NO: 1995) and ISIS Number 138678 (CATAAACATTTCCATCAGTG; herein incorporated as SEQ ID NO: 1996), both 5-10-5 2′-MOE gapmers with phosphorothioate backbones, were designed to target the human Dicer mRNA. These oligomeric compounds were used to transfect the A549, T-24, HepG2, HMEC, T47D, HuVEC, and MCF7 cell lines, as well as human primary dendritic cells, preadipocytes, differentiated adipocytes, and human spleen tissue, and the effects of treatment with the oligomeric compounds on phenotypic parameters, such as caspase activity and expression of markers of adipocyte differentiation (aP2, HSL, Glut4) was assessed as described in Examples 11 and 13, respectively.

Interestingly, treatment of T47D breast adenocarcinoma (p53 mutant) cells with the oligomeric compound ISIS 138648 targeting human Dicer was observed to result in a 41% increase in caspase activity. This phenotype is similar to the effect of treatment of T47D cells with oligomeric compound ISIS Number 328645 (SEQ ID NO: 554), targeting mir-124a-1 described in Example 11. It is believed that treatment of T47D cells with the oligomeric compound ISIS 138648 inhibits expression of human Dicer, which results in reduced production of mature miRNAs. Inadequate levels of miRNAs or inappropriately elevated levels of miRNA precursors may disrupt important cellular events, such as regulation of the cell cycle, and lead cells to trigger apoptotic pathways.

In adipocyte differentiation assays performed as described in Example 13, treatment of human white preadipocytes with ISIS Number 138648 targeting human Dicer was observed to result in decreased triglyceride production. An increase in triglyceride content is a well-established marker of adipocyte differentiation; treatment of adipocytes with oligomeric compound ISIS 138648 resulting in a decrease in triglyceride levels indicates an apparent inhibition of adipocyte differentiation. Thus, the oligomeric compound ISIS 138648 targeting human Dicer may be useful as a pharmaceutical agent with applications in the treatment, attenuation or prevention of obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases as well as in the maintenance of the undifferentiated, pluripotent phenotype of stem or precursor cells. The inhibition of expression of human Dicer by ISIS 138648 is believed to result in decreased production of miRNAs, and some of these miRNAs may be critical for proper regulation of the cell cycle (such as is predicted for the regulation of ERK5 by mir-143); treatment of preadipocytes with this inhibitor of human Dicer and the resulting decrease in production of mature miRNAs, as well as the concommitant accumulation of pre-miRNAs or pri-miRNAs may upset the balance between cellular proliferation and differentiation, predisposing cells to an undifferentiated state.

Example 37 Design of Additional Double-Stranded miRNA Mimics

As described supra, a reporter vector system employing, for example, the pGL3-bulge(×3) plasmid or the pGL3-mir-143 sensor plasmids can be used to assess the ability of miRNA mimics to bind target sites or to assess their effects on the expression of miRNAs, pre-miRNAs or pri-miRNAs. Various chemically modified miRNA mimics have been designed and synthesized for this purpose. The oligomeric compounds of the present invention can be designed to mimic a pri-miRNA, pre-miRNA or a single- or double-stranded miRNA while incorporating certain chemical modifications that alter one or more properties of the mimic, thus creating a construct with superior qualities over the endogenous precursor or miRNA.

In accordance with the present invention, a series of oligomeric compounds was designed and synthesized to mimic double-stranded miRNAs. In some embodiments, various oligomeric compounds representing the sense strand of the mir-143 miRNA, were synthesized, incorporating various chemically modified sugars and/or internucleoside linkages. Similarly, various oligomeric compounds representing the antisense strand complementary to the mir-143 miRNA were synthesized, incorporating various chemically modified sugars and/or internucleoside linkages. The antisense and sense oligomeric compounds designed to mimic mir-143 are shown in Table 68 and 69, respectively. All of the sugar moieties of the oligomeric compounds listed in Tables 68 and 69 are ribonucleotides unless otherwise indicated, and the 3′-terminal nucleosides each have a 3′-OH group unless otherwise specified. The sequences are written in the 5′ to 3′ direction. All antisense oligomeric compounds in Table 68 have the nucleotide sequence GAGCUACAGUGUUCAUCUCA (herein incorporated as SEQ ID NO: 1864). The sense oligomeric compounds in Table 69 have one of three nucleotide sequences which only differ in that there is a thymidine substitution in place of uridine in two of the sequences; these are: UGAGAUGAAGCACUGUAGCUC (herein incorporated as SEQ ID NO: 1088), UGAGATGAAGCACUGUAGCUC (herein incorporated as SEQ ID NO: 1088), and UGAGAUGAAGCACUGTAGCUC (herein incorporated as SEQ ID NO: 1088). In Tables 68 and 69, the column “Chemical modification” lists the general class and type of chemical modification for the respective oligomeric compounds. The column “Sequence” indicates the nucleobase sequence with symbols indicating sugar and linkage modifications. In the Sequence columns of Tables 68 and 69, internucleoside linkages are assumed to be phosphodiester unless otherwise indicated; phosphorothioate internucleoside linkages are indicated by “s” after the letter indicating the nucleobase (for example, “GsC” indicates a guanosine linked to a cytidine with a 3′,5′-phosphorothioate (PS) internucleoside linkage). Other symbols used to indicate sugar and linkage modifications in the Sequence columns of Tables 68 and 69 are as follows: “mc” indicates that the cytidine residue at the specified position is a 5-methylcytidine; replacement of the 2′-OH of the ribosyl sugar with a 2′-O-methoxyethyl (2′-MOE) is indicated by “e” after the letter indicating the nucleobase (for example, “GAe” indicates a guanosine linked to a 2′-MOE adenosine with a 3′,5′-phosphodiester internucleoside linkage); replacement or substitution of the 2′-OH of the ribosyl sugar with a 2-O-methyl (2′-OMe) is indicated by “m” after the letter indicating the nucleobase (for example, “CmA” indicates a 2′-O-methyl cytidine linked to an adenosine with a 3′,5′-phosphodiester internucleoside linkage); nucleosides having a 2′-Fluoro (2′-F) substituent group are indicated with a “f” after the letter indicating the nucleobase (for example, “GfAm” indicates a 2′-F guanosine linked to a 2′-O-Methyl-adenosine with a 3′,5′-phosphodiester internucleoside linkage); 4′-Thio (4′-S) residues are indicated by “4s” (for example, “GC4s” indicates a guanosine linked to a 4′-S cytidine with a 3′,5′-phosphodiester internucleoside linkage).

In the “Chemical modification” column of Tables 68 and 69, “unmodified” indicates a native strand. “Full” indicates a fully modified oligomeric compound where the chemical modification occurs at each nucleoside or internucleoside linkage. For example each nucleoside of the oligomeric compound could have a modified sugar selected from one of 4′-S, 2′-MOE, 2′-F, 2′-O-Methyl, LNA or ENA™ or could have uniformly modified internucleoside linkages such as uniform phosphorothioate internucleoside linkages.

In the “Chemical modification” column of Tables 68 and 69, “Alt” indicates that the nucleosides and or the internucleoside linkages have an alternating motif. The alternating motif can be the result of different sugar modifications that alternate (for example, 2′-ribose alternating with a 2′-modification other than ribose such as MOE, 2′-F or 2′-O-Methyl, or alternating fully modified sugars such as 2′-O-Methyl alternating with 2′-F), or can be the result of alternating internucleoside linkages (for example alternating phosphodiester and phosphorothioate internucleoside linkages). Oligomeric compounds having alternating modifications are described in the chemical modification column with the modification at the first 5′-nucleoside or the first internucleoside linkage at the 5′-end of the nucleoside listed first. For example, oligomeric compounds described as “Alt 2′-F/2′-OMe” have a 2′-F modified sugar at the 5′-terminal nucleoside with the next nucleoside having a 2′-F modified sugar and this alternating pattern is repeated through to the 3′-terminal nucleoside.

In the “Chemical modification” column of Tables 68 and 69, “gapmer” indicates that the oligomeric compound is divided into three distinct regions. The wings are the regions located externally at the 3′ and the 5′-end with the gap being the internal region. Gapmers can be the result of differences in linkage (PO vs. PS) or nucleoside (modified sugar moiety or heterocyclic base). Gapmers also include chimeric gapped oligomeric compounds such as when the wings and the gapped regions are all distinct one from each other. Examples of chemistries that can be used to prepare gapped oligomeric compounds include 2′-MOE, 2′-F, 2′-O-Methyl, LNA and ENA™.

In the “Chemical modification” column of Tables 68 and 69, “hemimer” indicates an oligomeric compound that has two distinct regions resulting from differences in the nucleoside or the internucleoside linkage or both. Examples include oligomeric compounds having two regions wherein one region has modified internucleoside linkages such as PS or modified sugar moieties such as 2′-MOE, 2′-F, 2′-O-Methyl, LNA and ENA™.

In the “Chemical modification” column of Tables 68 and 69, “blockmer” indicates an oligomeric compound that has at least one block of modified nucleosides or internucleoside linkages that are located internally. The blocks are generally from two to about five nucleosides in length and are not located at one of the ends as that could be a hemimer. Examples of blockmers include oligomeric compounds having from two to about five internally modified nucleosides such as 2′-MOE, 2′-F, 2′-O-Methyl, LNA and ENA™.

In the “Chemical modification” column of Tables 68 and 69, “point modification” indicates an oligomeric compound having a single modified nucleoside located in the oligomeric compound at any position.

TABLE 68 Antisense oligomeric compounds mimicking mir-143 SEQ ISIS ID Chemical NO: NO modification Sequence 348173 1864 Unmodified GAGCUACAGUGCUUCAUCUCA 348187 1864 Full PS GsAsGsCsUsAsCsAsGsUsGsCsUsUsCsAsUsCsUsCsA 362972 1864 Alt ribose/2′- GAeGCeUAeCAeGUeGCeUUeCAeUCeUCeA MOE 366179 1864 Alt ribose/2′- GAmGCmUAmCAmGUmGCmUUmCAmUCmUCmA OMe 366181 1864 Alt 2′- GmAGmCUmACmAGmUGmCUmUCmAUmCUmCAm OMe/ribose 366182 1864 Full 2′-OMe GmAmGmCmUmAmCmAmGmUmGmCmUmUmCmAmUmCmUmCmAm 366188 1864 2′-MOE 3-15-3 GeAeGeCUACAGUGCUUCAUCUeCeAe gapmer 366189 1864 Full 2′-MOE GeAeGeCeUeAeCeAeGeUeGeCeUeUeCeAeUeCeUeCeAe 366190 1864 Alt 2′- GeAGeCUeACeAGeUGeCUeUCeAUeCUeCAe MOE/ribose 366198 1864 Alt 2′-F/2′-OMe  GfAmGfCmUfAmCfAmGfUmGfCmUfUmCfAmUfCmUfCmAf

TABLE 69 Sense oligomeric compounds mimicking mir-143 SEQ ISIS ID Chemical NO: NO modification Sequence 348201 1088 Unmodified UGAGAUGAAGCACUGUAGCUC 342199 220 Unmodified UGAGAUGAAGCACUGUAGCUCA 348215 1088 Full PS UsGsAsGsAsUsGsAsAsGsCsAsCsUsGsUsAsGsCsUsC 366175 1088 PO/PS/PO UGAGAUGAAGsCsAsCsUsGUAGCUC gapmer 366176 1088 5′ PS hemimer UsGsAsGsAsUGAAGCACUGUAGCUC 366177 1088 3′ PS hemimer UGAGAUGAAGCACUGUsAsGsCsUsC 366178 1088 Alt 2′- UmGAmGAmUGmAAmGCmACmUGmUAmGCmUCm OMe/ribose 366180 1088 Alt UGmAGmAUmGAmAGmCAmCUmGUmAGmCUmC ribose/2′OMe 366183 1088 2′-OMe UGAGAUmGmAAGmCmACUGUAGCmUmCm blockmer 366184 1088 2′-OMe UGAGAUGAmAmGCAmCmUGUAGCmUmCm blockmer 366185 1088 2′-MOE UGAGAUGAAGCAeCeUGUAGCUC blockmer 366186 1088 2′-MOE UGAGeAeUeGAAGCACUGUAGCUC blockmer 366187 1088 2′-MOE UGAGAUGAAGCACUGUeAeGeCUC blockmer 366191 1088 4′s gapmer U4sGAGAUGAAGCACUGUAGC4sU4sC4s 366192 1088 4′s 2 ′-OMe U4sGAGAUGAAGCACUGUAGCmUmCm gapmer 366193 1088 2′-F blockmer UGfAfGfAfUfGfAfAfGCACUGUAGCUC 366194 1088 LNA blockmer UGAGlAlUlGAAGCACUGUAGCUC 366195 1088 LNA blockmer UGAGAUGAAGCACUGUlAlGlCUC 366196 1088 LNA blockmer UGAGAUGAAGCAlClUGUAGCUC 366197 1088 Alt 2′- UmGfAmGfAmUfGmAfAmGfCmAfCmUfGmUfAmGfCmUfCm OMe/2′F 366209 1088 LNA blockmer UGAGlAlTlGAAGCACUGUAGCUC 366210 1088 LNA blockmer UGAGAUGAAGCACUGTlAlGlCUC 366211 1088 LNA point UGAGAUGAAGCAl^(m)ClUGUAGCUC modification 

Oligomeric compounds representing mimics of the antisense and the sense strands of a double-stranded miRNA can be hybridized, and various combinations of synthetic, modified or unmodified double-stranded oligomeric compounds, each representing a double-stranded miRNA mimic, may be formed. With the various chemical modifications, many permutations of such double-stranded miRNA mimics can be achieved. These double-stranded oligomeric compounds can be blunt-ended or can comprise two strands differing in length such that the resulting double-stranded oligomeric compound has a 3′- and/or a 5′-overhang of one to five nucleotides on either the sense and/or antisense strands. The compounds can be analyzed for their ability to mimic miRNAs, pre-miRNAs, or pri-miRNAs and to bind to nucleic acid targets (for example, RNA transcripts, mRNAs, reporter constructs), for their effects on miRNA, pre-miRNA, or pri-miRNA expression levels by quantitative real-time PCR, or they can be used in other in vivo or in vitro phenotypic assays to investigate the role of miRNAs in regulation of downstream nucleic acid targets, as described in other examples herein. These oligomeric compounds of the present invention may disrupt pri-miRNA and/or pre-miRNA structures, and sterically hinder cleavage by Drosha-like and/or Dicer-like Rnase III enzymes, respectively. Oligomeric compounds capable of binding to the mature miRNA are also predicted to prevent the RISC-mediated binding of a miRNA to its mRNA target, either by cleavage or steric occlusion of the miRNA.

In some embodiments, HeLa cells transiently expressing the pGL3-mir-143 sensor reporter vector and the pRL-CMV Renilla luciferase plasmids, as described in Example 27, were also treated with double-stranded oligomeric compounds produced by hybridizing an antisense oligomeric compound from Table 68 with a sense oligomeric compound from Table 69, as described herein. HeLa cells were routinely cultured and passaged and on the day before transfection, the HeLa cells were seeded onto 96-well plates 3,000 cells/well. Cells were transfected according to standard published procedures with plasmids using 2 μg Lipofectamine™ 2000 Reagent (Invitrogen) per μg of plasmid DNA, or, when transfecting double-stranded oligomeric compounds, 1.25 μg of Lipofectamine™ 2000 Reagent was used per 100 nM oligonucleotide. Cells were treated at 10 nM and 100 nM with the double-stranded oligomeric compound mimics. A double-stranded oligomeric compound representing a 10-base mismatched sequence antisense to the unrelated PTP1B mRNA, composed of ISIS Number 342427 (SEQ ID NO: 863) hybridized to its perfect complement ISIS Number 342430 (SEQ ID NO: 864) was used as a negative control (“ds-Control”). The pGL3-mir-143 sensor reporter plasmid was transfected at 0.025 μg per well. The luciferase signal in each well was normalized to the Renilla luciferase (RL) activity produced from the co-transfected pRL-CMV plasmid, which was transfected at 2.5 μg per well. In accordance with methods described in Example 12 and 27, a luciferase assay was performed 48-hours after transfection. Briefly, cells were lysed in passive lysis buffer (PLB; Promega), and 20 ul of the lysate was then assayed for RL activity using a Dual Luciferase Assay kit (Promega) according to the manufacturer's protocol. The results below are an average of two trials and are presented as percent pGL3-Control luciferase expression normalized to pRL-CMV expression (RL). The data are shown in Table 70.

TABLE 70 Luciferase assays showing effects of double-stranded compounds mimicking mir-143 ISIS Numbers luciferase expression hybridized to (% lucif.only control) form ds compound 10 nM treatment 100 nM treatment pGL3-mir-143 sensor + 79.4 94.1 pRL-CMV only pGL3-mir-143 sensor + 120.6 105.9 pRL-CMV only 342430 + 342427 75.0 86.1 ds-Control 348215 + 348173 23.1 37.5 348215 + 362972 28.6 32.4 366175 + 348173 20.0 25.0 366175 + 362972 56.9 33.4 366176 + 348173 42.6 30.0 366176 + 362972 63.4 98.5 366177 + 348173 35.7 33.6 366177 + 362972 32.8 29.1 366183 + 348173 29.2 24.5 366183 + 362972 54.3 36.8 366184 + 348173 35.6 27.7 366184 + 362972 47.3 31.9 366185 + 348173 22.2 18.5 366185 + 362972 27.2 28.7 366186 + 348173 34.8 26.8 366186 + 362972 50.2 60.8 366187 + 348173 34.6 32.4 366187 + 362972 25.5 27.9 366209 + 348173 112.9 85.4 366209 + 362972 111.3 97.5 366210 + 348173 37.1 28.2 366210 + 362972 51.8 41.1 366211 + 348173 32.1 28.7 366211 + 362972 46.6 36.7 366193 + 348173 20.0 17.6 366193 + 362972 24.4 22.6 366191 + 348173 27.3 26.9 366191 + 362972 37.5 25.8 366192 + 348173 22.3 27.9 366192 + 362972 28.9 25.7 366197 + 348173 37.0 22.2 366197 + 362972 42.0 32.7 366197 + 366198 30.2 28.7 366178 + 348173 75.0 74.0 366178 + 362972 98.6 104.0 366178 + 366179 63.5 75.4 366178 + 366181 74.1 70.6 366180 + 366179 97.0 38.5 366180 + 366181 43.5 50.2 pGL3-mir-143 sensor + 100.0 112.9 pRL-CMV only 342430 + 342427 81.2 165.9 ds-Control 348201 + 348187 44.0 55.4 348201 + 366182 138.9 89.2 348201 + 366179 76.2 68.5 348201 + 366181 92.2 340.0 348201 + 362972 65.2 67.3 348201 + 366198 47.3 58.8 342199 + 348173 40.3 122.0 342199 + 348187 91.3 55.5 342199 + 366182 47.4 84.1 342199 + 366179 76.5 45.9 342199 + 366181 86.1 34.2 342199 + 362972 50.8 78.7 342199 + 366189 26.7 45.2 342199 + 366190 93.0 37.9 342199 + 366198 52.5 45.5

From these data, it was observed that treatment of HeLa cells expressing the pGL3-mir-143 sensor reporter vector with many of the double-stranded oligomeric compounds mimicking mir-143 at both the 10 nM and 100 nM concentrations resulted in inhibition of luciferase activity. For example, the double stranded oligomeric compounds comprising ISIS Number 348173 as an unmodified antisense strand in combination with ISIS Number 366177 (a hemimer with phosphorothioate modified residues at the 3′ end) or ISIS Number 366185 (a 2′-MOE blockmer) as the modified sense strand resulted in significant reductions in luciferase activity. Furthermore, double stranded oligomeric compounds comprising, as the antisense strand, either ISIS Number 366189 (a fully modified 2′-MOE compound) or ISIS Number 366198 (with alternating 2′-Fluoro and 2′-O-Methyl residues), in combination with ISIS Number 342199 as the unmodified sense strand resulted in significant reductions in luciferase activity, indicating that these compounds are effective mir-143 mimics. Taken with the previous observations that the mir-143 miRNA is involved in adipocyte differentiation, these double-stranded mir-143 mimics may be useful as therapeutic agents with applications in the treatment, attenuation or prevention of obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases as well as having potential applications in the maintenance of the pluripotent phenotype of stem or precursor cells.

Example 38 Design of Oligomeric Compounds Targeting Pri-miRNAs

As described above, mature miRNAs originate from pri-miRNAs, which are believed to be processed into pre-miRNAs by the Drosha RNase III enzyme, and subsequently exported from the nucleus to the cytoplasm, where the pre-miRNAs are processed by human Dicer into double-stranded intermediates resembling siRNAs, which are then processed into mature miRNAs.

Some oligomeric compounds of the present invention are believed to bind to pri-miRNA molecules and interfere with their processing into a mature miRNA. These oligomeric compounds were observed to affect a decrease in expression levels of mature miRNA, presumably due, at least in part, to steric interference with their processing into mature miRNAs by human Dicer. Furthermore, as described above, some oligomeric compounds of the present invention have been observed to affect an increase in expression levels of pri-miRNAs; it is believed that the decrease in levels of mature miRNAs cells treated with these oligomeric compounds may trigger a feedback mechanism that signals these cells to increase production of the pri-miRNA molecule. This increase may be the result, at least in part, of a stimulation of transcription of the pri-miRNAs in response to the decrease in mature miRNAs. Not mutually exclusive with the processing interference and the feedback mechanisms is the possibility that treatment with oligomeric compounds could stimulate the activity of an RNA-dependent RNA polymerase (RdRP) that amplifies pre-miRNAs or pri-miRNAs.

In one embodiment, several nested series of single-stranded oligomeric compounds, 15-nucleotides in length, composed of 2′-methoxyethoxy (2′-MOE) modified nucleotides and phosphorothioate (P═S) internucleoside linkages throughout the compound, were designed and synthesized to target several pri-miRNAs, to test the effects of these compounds on the expression levels of small non-coding RNAs. These compounds are shown in Table 71, below. “Pri-miRNA” indicates the particular pri-miRNA which contains the miRNA that the oligomeric compound was designed to target. The “Region” column describes the general region of the pri-miRNA that is being targeted. The following features of the stem loop structures of pri-miRNA were targeted: 1) “5′-stem side mir start” means the 5′-stem side at the 5′-end of the sequence representing the mature miRNA, with the oligomeric compounds targeting and spanning sequences completely outside of the mature miRNA to completely within it; 2) “5′-stem side mir end” means the 5′-stem side at the 3′-end of the sequence representing the mature miRNA, with the oligomeric compounds targeting and spanning sequences completely within the mature miRNA to spanning and extending beyond the 3′-end of it; 3) “loop start” means the 5′-side of the loop region; 4) “loop end” means with the oligomeric compounds targeting and ending at the 3′-side of the loop region; 5) “3′-stem side mir start” means the 3′-stem side at the 5′-end of the sequence representing the mature miRNA, with the oligomeric compounds targeting and completely within the mature miRNA to a few nucleotides outside of it; 6) “3′-stem side mir end” means the 3′-stem side at the 3′-end of the sequence representing the mature miRNA, with the oligomeric compounds targeting and spanning sequences completely within the mature miRNA to completely outside of it.

TABLE 71 Uniform 2′-MOE oligomeric compounds targeting pri-miRNAs SEQ ID pri-miRNA Region Isis # Sequence NO: mir-182 mir-182 5′-stem side mir start 366888 AAACGGGGGGAGGCA 1997 mir-182 mir-182 5′-stem side mir start 366889 GCCAAAAACGGGGGG 1998 mir-182 mir-182 5′-stem side mir start 366890 ATTGCCAAAAACGGG 1999 mir-182 mir-182 5′-stem side mir start 366891 ACCATTGCCAAAAAC 2000 mir-182 mir-182 5′-stem side mir start 366892 TCTACCATTGCCAAA 2001 mir-182 mir-182 5′-stem side mir end 366893 TGTGAGTTCTACCAT 2002 mir-182 mir-182 5′-stem side mir end 366894 CAGTGTGAGTTCTAC 2003 mir-182 mir-182 5′-stem side mir end 366895 CACCAGTGTGAGTTC 2004 mir-182 mir-182 5′-stem side mir end 366896 CCTCACCAGTGTGAG 2005 mir-182 mir-182 loop start 366897 TCCTGTTACCTCACC 2006 mir-182 mir-182 loop start 366898 GATCCTGTTACCTCA 2007 mir-182 mir-182 loop start 366899 CGGATCCTGTTACCT 2008 mir-182 mir-182 loop end 366900 TGTTACCTCACCAGT 2009 mir-182 mir-182 loop end 366901 CCTGTTACCTCACCA 2010 mir-182 mir-182 loop end 366902 ATCCTGTTACCTCAC 2011 mir-182 mir-182 loop end 366903 GGATCCTGTTACCTC 2012 mir-182 mir-182 loop end 366904 CCGGATCCTGTTACC 2013 mir-182 mir-182 3′-stem side mir start 366905 GAACCACCGGATCCT 2014 mir-182 mir-182 3′-stem side mir start 366906 CTAGAACCACCGGAT 2015 mir-182 mir-182 3′-stem side mir start 366907 AGTCTAGAACCACCG 2016 mir-182 mir-182 3′-stem side mir start 366908 GCAAGTCTAGAACCA 2017 mir-182 mir-182 3′-stem side mir end 366909 ATAGTTGGCAAGTCT 2018 mir-182 mir-182 3′-stem side mir end 366910 CGCCCCATAGTTGGC 2019 mir-182 mir-182 3′-stem side mir end 366911 CCTCGCCCCATAGTT 2020 mir-182 mir-182 3′-stem side mir end 366912 AGTCCTCGCCCCATA 2021 mir-182 mir-182 3′-stem side mir end 366913 CTGAGTCCTCGCCCC 2022 mir-216 mir-216 5′-stem side mir start 366914 AAGCCAACTCACAGC 2023 mir-216 mir-216 5′-stem side mir start 366915 AGATTAAGCCAACTC 2024 mir-216 mir-216 5′-stem side mir start 366916 CTGAGATTAAGCCAA 2025 mir-216 mir-216 5′-stem side mir start 366917 CAGCTGAGATTAAGC 2026 mir-216 mir-216 5′-stem side mir start 366918 TGCCAGCTGAGATTA 2027 mir-216 mir-216 5′-stem side mir end 366919 TCACAGTTGCCAGCT 2028 mir-216 mir-216 5′-stem side mir end 366920 ATCTCACAGTTGCCA 2029 mir-216 mir-216 5′-stem side mir end 366921 AACATCTCACAGTTG 2030 mir-216 mir-216 5′-stem side mir end 366922 ATGAACATCTCACAG 2031 mir-216 mir-216 loop start 366923 ATTGTATGAACATCT 2032 mir-216 mir-216 loop start 366924 GGATTGTATGAACAT 2033 mir-216 mir-216 loop start 366925 AGGGATTGTATGAAC 2034 mir-216 mir-216 loop end 366926 TGTATGAACATCTCA 2035 mir-216 mir-216 loop end 366927 TGAGGGATTGTATGA 2036 mir-216 mir-216 3′-stem side mir start 366928 ACTGTGAGGGATTGT 2037 mir-216 mir-216 3′-stem side mir start 366929 ACCACTGTGAGGGAT 2038 mir-216 mir-216 3′-stem side mir start 366930 GAGACCACTGTGAGG 2039 mir-216 mir-216 3′-stem side mir start 366931 CCAGAGACCACTGTG 2040 mir-216 mir-216 3′-stem side mir end 366932 CATAATCCCAGAGAC 2041 mir-216 mir-216 3′-stem side mir end 366933 GTTTAGCATAATCCC 2042 mir-216 mir-216 3′-stem side mir end 366934 TCTGTTTAGCATAAT 2043 mir-216 mir-216 3′-stem side mir end 366935 TGCTCTGTTTAGCAT 2044 mir-216 mir-216 3′-stem side mir end 366936 AATTGCTCTGTTTAG 2045 mir-143 mir-143 5′-stem side mir start 366937 AGGCTGGGAGACAGG 2046 mir-143 mir-143 5′-stem side mir start 366938 ACCTCAGGCTGGGAG 2047 mir-143 mir-143 5′-stem side mir start 366939 TGCACCTCAGGCTGG 2048 mir-143 mir-143 5′-stem side mir start 366940 CACTGCACCTCAGGC 2049 mir-143 mir-143 5′-stem side mir start 366941 CAGCACTGCACCTCA 2050 mir-143 mir-143 5′-stem side mir end 366942 AGAGATGCAGCACTG 2051 mir-143 mir-143 5′-stem side mir end 366943 ACCAGAGATGCAGCA 2052 mir-143 mir-143 5′-stem side mir end 366944 CTGACCAGAGATGCA 2053 mir-143 mir-143 5′-stem side mir end 366945 CAACTGACCAGAGAT 2054 mir-143 mir-143 loop start 366946 CAGACTCCCAACTGA 2055 mir-143 mir-143 loop start 366947 CTCAGACTCCCAACT 2056 mir-143 mir-143 loop start 366948 ATCTCAGACTCCCAA 2057 mir-143 mir-143 loop end 366949 AACTGACCAGAGATG 2058 mir-143 mir-143 loop end 366950 CCAACTGACCAGAGA 2059 mir-143 mir-143 loop end 366951 TCCCAACTGACCAGA 2060 mir-143 mir-143 loop end 366952 ACTCCCAACTGACCA 2061 mir-143 mir-143 3′-stem side mir start 366953 TTCATCTCAGACTCC 2062 mir-143 mir-143 3′-stem side mir start 366954 TGCTTCATCTCAGAC 2063 mir-143 mir-143 3′-stem side mir start 366955 CAGTGCTTCATCTCA 2064 mir-143 mir-143 3′-stem side mir end 366956 TGAGCTACAGTGCTT 2065 mir-143 mir-143 3′-stem side mir end 366957 TCTTCCTGAGCTACA 2066 mir-143 mir-143 3′-stem side mir end 366958 CTCTCTTCCTGAGCT 2067 mir-143 mir-143 3′-stem side mir end 366959 CTTCTCTCTTCCTGA 2068 mir-143 mir-143 3′-stem side mir end 366960 CAACTTCTCTCTTCC 2069 mir-23b mir-23b 5′-stem side mir start 366961 AGCAGCCAGAGCACC 2070 mir-23b mir-23b 5′-stem side mir start 366962 ACCCAAGCAGCCAGA 2071 mir-23b mir-23b 5′-stem side mir start 366963 GGAACCCAAGCAGCC 2072 mir-23b mir-23b 5′-stem side mir start 366964 CCAGGAACCCAAGCA 2073 mir-23b mir-23b 5′-stem side mir start 366965 ATGCCAGGAACCCAA 2074 mir-23b mir-23b 5′-stem side mir end 366966 AATCAGCATGCCAGG 2075 mir-23b mir-23b 5′-stem side mir end 366967 ACAAATCAGCATGCC 2076 mir-23b mir-23b 5′-stem side mir end 366968 GTCACAAATCAGCAT 2077 mir-23b mir-23b 5′-stem side mir end 366969 TAAGTCACAAATCAG 2078 mir-23b mir-23b loop start 366970 AATCTTAAGTCACAA 2079 mir-23b mir-23b loop start 366971 TTAATCTTAAGTCAC 2080 mir-23b mir-23b loop start 366972 TTTTAATCTTAAGTC 2081 mir-23b mir-23b loop end 366973 CTTAAGTCACAAATC 2082 mir-23b mir-23b loop end 366974 ATCTTAAGTCACAAA 2083 mir-23b mir-23b loop end 366975 TAATCTTAAGTCACA 2084 mir-23b mir-23b loop end 366976 TTTAATCTTAAGTCA 2085 mir-23b mir-23b loop end 366977 ATTTTAATCTTAAGT 2086 mir-23b mir-23b 3′-stem side mir start 366978 TGTGATTTTAATCTT 2087 mir-23b mir-23b 3′-stem side mir start 366979 CAATGTGATTTTAAT 2088 mir-23b mir-23b 3′-stem side mir start 366980 TGGCAATGTGATTTT 2089 mir-23b mir-23b 3′-stem side mir start 366981 CCCTGGCAATGTGAT 2090 mir-23b mir-23b 3′-stem side mir end 366982 TGGTAATCCCTGGCA 2091 mir-23b mir-23b 3′-stem side mir end 366983 GTTGCGTGGTAATCC 2092 mir-23b mir-23b 3′-stem side mir end 366984 GTGGTTGCGTGGTAA 2093 mir-23b mir-23b 3′-stem side mir end 366985 GTCGTGGTTGCGTGG 2094 mir-23b mir-23b 3′-stem side mir end 366986 AAGGTCGTGGTTGCG 2095 mir-203 mir-203 5′-stem side mir start 366987 GACCCAGCGCGCGAG 2096 mir-203 mir-203 5′-stem side mir start 366988 CACTGGACCCAGCGC 2097 mir-203 mir-203 5′-stem side mir start 366989 AACCACTGGACCCAG 2098 mir-203 mir-203 5′-stem side mir start 366990 AAGAACCACTGGACC 2099 mir-203 mir-203 5′-stem side mir start 366991 GTTAAGAACCACTGG 2100 mir-203 mir-203 5′-stem side mir end 366992 TTGAACTGTTAAGAA 2101 mir-203 mir-203 5′-stem side mir end 366993 CTGTTGAACTGTTAA 2102 mir-203 mir-203 5′-stem side mir end 366994 GAACTGTTGAACTGT 2103 mir-203 mir-203 5′-stem side mir end 366995 ACAGAACTGTTGAAC 2104 mir-203 mir-203 loop start 366996 AATTGCGCTACAGAA 2105 mir-203 mir-203 loop start 366997 ACAATTGCGCTACAG 2106 mir-203 mir-203 loop start 366998 TCACAATTGCGCTAC 2107 mir-203 mir-203 loop end 366999 TACAGAACTGTTGAA 2108 mir-203 mir-203 loop end 367000 GCTACAGAACTGTTG 2109 mir-203 mir-203 loop end 367001 GCGCTACAGAACTGT 2110 mir-203 mir-203 loop end 367002 TTGCGCTACAGAACT 2111 mir-203 mir-203 3′-stem side mir start 367003 TTTCACAATTGCGCT 2112 mir-203 mir-203 3′-stem side mir start 367004 ACATTTCACAATTGC 2113 mir-203 mir-203 3′-stem side mir start 367005 TAAACATTTCACAAT 2114 mir-203 mir-203 3′-stem side mir start 367006 TCCTAAACATTTCAC 2115 mir-203 mir-203 3′-stem side mir end 367007 CTAGTGGTCCTAAAC 2116 mir-203 mir-203 3′-stem side mir end 367008 CCGGGTCTAGTGGTC 2117 mir-203 mir-203 3′-stem side mir end 367009 CCGCCGGGTCTAGTG 2118 mir-203 mir-203 3′-stem side mir end 367010 CGCCCGCCGGGTCTA 2119 mir-203 mir-203 3′-stem side mir end 367011 CCGCGCCCGCCGGGT 2120 mir-21 mir-21 5′-stem side mir start 367012 GCTACCCGACAAGGT 2121 mir-21 mir-21 5′-stem side mir start 367013 AAGCTACCCGACAAG 2122 mir-21 mir-21 5′-stem side mir start 367014 GATAAGCTACCCGAC 2123 mir-21 mir-21 5′-stem side mir start 367015 TCTGATAAGCTACCC 2124 mir-21 mir-21 5′-stem side mir start 367016 CAGTCTGATAAGCTA 2125 mir-21 mir-21 5′-stem side mir end 367017 TCAACATCAGTCTGA 2126 mir-21 mir-21 5′-stem side mir end 367018 CAGTCAACATCAGTC 2127 mir-21 mir-21 5′-stem side mir end 367019 CAACAGTCAACATCA 2128 mir-21 mir-21 5′-stem side mir end 367020 ATTCAACAGTCAACA 2129 mir-21 mir-21 loop start 367021 GCCATGAGATTCAAC 2130 mir-21 mir-21 loop start 367022 TTGCCATGAGATTCA 2131 mir-21 mir-21 loop start 367023 TGTTGCCATGAGATT 2132 mir-21 mir-21 loop end 367024 TTCAACAGTCAACAT 2133 mir-21 mir-21 loop end 367025 GATTCAACAGTCAAC 2134 mir-21 mir-21 loop end 367026 GAGATTCAACAGTCA 2135 mir-21 mir-21 loop end 367027 ATGAGATTCAACAGT 2136 mir-21 mir-21 loop end 367028 CCATGAGATTCAACA 2137 mir-21 mir-21 3′-stem side mir start 367029 GTGTTGCCATGAGAT 2138 mir-21 mir-21 3′-stem side mir start 367030 CTGGTGTTGCCATGA 2139 mir-21 mir-21 3′-stem side mir start 367031 CGACTGGTGTTGCCA 2140 mir-21 mir-21 3′-stem side mir start 367032 CATCGACTGGTGTTG 2141 mir-21 mir-21 3′-stem side mir end 367033 GACAGCCCATCGACT 2142 mir-21 mir-21 3′-stem side mir end 367034 ATGTCAGACAGCCCA 2143 mir-21 mir-21 3′-stem side mir end 367035 AAATGTCAGACAGCC 2144 mir-21 mir-21 3′-stem side mir end 367036 CAAAATGTCAGACAG 2145 mir-221 mir-221 5′-stem side mir start 367037 CATGCCCCAGACCTG 2146 mir-221 mir-221 5′-stem side mir start 367038 AGGTTCATGCCCCAG 2147 mir-221 mir-221 5′-stem side mir start 367039 GCCAGGTTCATGCCC 2148 mir-221 mir-221 5′-stem side mir start 367040 TATGCCAGGTTCATG 2149 mir-221 mir-221 5′-stem side mir start 367041 TTGTATGCCAGGTTC 2150 mir-221 mir-221 5′-stem side mir end 367042 ATCTACATTGTATGC 2151 mir-221 mir-221 5′-stem side mir end 367043 GAAATCTACATTGTA 2152 mir-221 mir-221 5′-stem side mir end 367044 ACAGAAATCTACATT 2153 mir-221 mir-221 5′-stem side mir end 367045 AACACAGAAATCTAC 2154 mir-221 mir-221 loop start 367046 CTGTTGCCTAACGAA 2155 mir-221 mir-221 loop start 367047 AGCTGTTGCCTAACG 2156 mir-221 mir-221 loop start 367048 GTAGCTGTTGCCTAA 2157 mir-221 mir-221 loop end 367049 GAACACAGAAATCTA 2158 mir-221 mir-221 loop end 367050 ACGAACACAGAAATC 2159 mir-221 mir-221 loop end 367051 TAACGAACACAGAAA 2160 mir-221 mir-221 loop end 367052 CCTAACGAACACAGA 2161 mir-221 mir-221 loop end 367053 TGCCTAACGAACACA 2162 mir-221 mir-221 3′-stem side mir start 367054 AATGTAGCTGTTGCC 2163 mir-221 mir-221 3′-stem side mir start 367055 GACAATGTAGCTGTT 2164 mir-221 mir-221 3′-stem side mir start 367056 GCAGACAATGTAGCT 2165 mir-221 mir-221 3′-stem side mir end 367057 AAACCCAGCAGACAA 2166 mir-221 mir-221 3′-stem side mir end 367058 AGCCTGAAACCCAGC 2167 mir-221 mir-221 3′-stem side mir end 367059 GGTAGCCTGAAACCC 2168 mir-221 mir-221 3′-stem side mir end 367060 CCAGGTAGCCTGAAA 2169 mir-221 mir-221 3′-stem side mir end 367061 TTTCCAGGTAGCCTG 2170

These modified oligomeric compounds targeting pri-miRNAs can be transfected into preadipocytes or other undifferentiated cells, which are then induced to differentiate, and it can be determined whether these modified oligomeric compounds act to inhibit or promote cellular differentiation. These compounds can be transfected into differentiating adipocytes and their effects on expression levels of the pri-miRNA molecules assessed in pre-adipocytes vs. differentiated adipocytes. By using a primer/probe set specific for the pri-miRNA or the pre-miRNA, real-time RT-PCR methods can be used to determine whether modified oligomeric compounds targeting pri-miRNAs can affect the expression or processing of the mature miRNAs from the pri-miRNA or pre-miRNA molecules.

Example 39 Effects of Oligomeric Compounds Targeting miRNAs in the Immune Response

To investigate the role of miRNAs in the immune response, oligomeric compounds of the present invention targeting miRNAs were tested for their effects upon lipopolysaccharide (LPS)-activated primary murine macrophages. Macrophages participate in the immune response, for example, in the recognition and phagocytosis of microorganisms, including bacteria. Interferon-gamma (IFN-gamma) released by helper T cells is one type of signal required for macrophage activation, and LPS can serve as an additional stimulus. LPS is a component of the gram-negative bacterial cell wall and acts as an agonist for toll-like receptor 4 (TLR4), the primary LPS receptor expressed by macrophages. The proinflammatory cytokines interleukin-12 (IL-12) and interleukin-6 (IL-6) are induced by LPS treatment of macrophages, thus the expression of the mRNAs encoding these cytokines was used to evaluate the response of macrophages to LPS following treatment with oligomeric compounds targeting miRNAs.

Macrophages were isolated as follows. Female C57B1/6 mice (Charles River Laboratories, Wilmington, Mass.) were injected intraperitoneally with 1 ml 3% thioglycollate broth (Sigma-Aldrich, St. Louis, Mo.), and peritoneal macrophage cells were isolated by peritoneal lavage 4 days later. The cells were plated in 96-well plates at 40,000 cells/well for one hour in serum-free RPMI adjusted to contain 10 mM HEPES (Invitrogen Life Technologies, Carlsbad, Calif.), allowed to adhere, then non-adherent cells were washed away and the media was replaced with RPMI containing 10 mM HEPES, 10% FBS and penicillin/streptomycin (“complete” RPMI; Invitrogen Life Technologies, Carlsbad, Calif.).

Oligomeric compounds were introduced into the cells using the non-liposomal transfection reagent FuGENE 6 Transfection Reagent (Roche Diagnostics Corp., Indianapolis, Ind.). Oligomeric compound was mixed with FuGENE 6 in 1 mL of serum-free RPMI to achieve a concentration of 10 μL FuGENE per 1000 nM oligomeric compound. The oligomeric compound/FuGENE complex was allowed to form at room temperature for 20 minutes. This mixture was diluted to the desired concentration of oligomeric compound by the addition of the appropriate volume of complete RPMI. The final ratio of FuGENE 6 to oligomeric compound was 1 μL of FuGENE 6 per 100 nM oligomeric compound. A volume of 100 μL of oligomeric compound/FuGENE/RPMI was added to each well of the 96-well plate in which the macrophages were cultured. Each oligomeric compound treatment was repeated in triplicate.

Following oligomeric compound treatment, cells were stimulated with LPS. Cells were cultured in the presence of the transfection complex for approximately 24 to 28 hours at 37° C. and 5% CO₂, after which the medium containing the transfection complex was removed from the cells, and complete RPMI containing 100 ng/mL LPS (Sigma-Aldrich Corp., St. Louis, Mo.) was added to the cells for a period of approximately 24 hours. Control samples included (1) cells receiving no oligomeric compound, stimulated with LPS and (2) cells receiving neither oligomeric compound nor LPS treatment.

In another embodiment, following oligomeric compound treatment, cells were first activated by IFN-gamma, to amplify the response to LPS. Cells were cultured in the presence of the transfection complex for approximately 24 hours at 37° C. and 5% CO₂, at which point the medium containing the transfection complex was removed from the cells, and complete RPMI containing 100 ng/mL recombinant mouse IFN-gamma (R&D Systems, Minneapolis, Minn.) was added to the cells. After the 4 hour treatment with INF-gamma, cells were treated with 100 ng/mL LPS for approximately 24 hours. Control samples included (1) cells receiving no oligomeric compound, stimulated with LPS and (2) cells receiving neither oligomeric compound nor LPS treatment.

Oligomeric compounds used as negative controls included ISIS 129690 (SEQ ID NO: 907), a universal scrambled control; ISIS 342673 (SEQ ID NO: 758), an oligomeric compound containing 15 mismatches with respect to the mature mir-143 miRNA; ISIS 342683 (SEQ ID NO: 790), an oligomeric compound representing the scrambled nucleotide sequence of an unrelated PTP1B antisense oligonucleotide; and ISIS 289606 (CCTTCCCTGAAGGTTCCTCC, incorporated herein as SEQ ID NO: 863), an oligomeric compound representing the scrambled nucleotide sequence of an unrelated PTP1B antisense oligonucleotide. ISIS 289606 is uniformly composed of 2′-MOE nucleotides, with phosphorothioate internucleoside linkages throughout the compound. All cytidines are 5-methyl cytidines. Used as a positive control was ISIS 229927 (CCACATTGAGTTTCTTTAAG, incorporated herein as SEQ ID NO: 2171), targeting the mouse toll-like receptor 4 (TLR4) mRNA, which is the primary LPS receptor on macrophages. ISIS 229927 is a chimeric oligomeric compound (“gapmer”) composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five nucleotide “wings,” wherein the wings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides. Internucleoside linkages are phosphorothioate throughout the compound, and all cytidines are 5-methylcytidines. Treatments with control oligomeric compounds were performed as described for oligomeric compounds targeting miRNAs.

Following the 24 hour treatment with LPS, the cells were lysed and RNA was isolated using the RNEASY 96™ kit, as described herein. mRNA expression was quantitated by real-time PCR, performed as described herein, using primer and probe sets to amplify and quantitate TLR4, IL-12 and IL-6 mRNA expression levels. Primers and probe for TLR4, designed using GenBank Accession number NM_(—)021297.1, were: forward primer, 5′-CATGGAACACATGGCTGCTAA-3′ (SEQ ID NO: 2172), reverse primer, 5′-GGAAAGGAAGGTGTCAGTGCTACT-3′ (SEQ ID NO: 2173), probe 5′-FAM-TAGCATGGACCTTACCGGGCAGAAGG-TAMRA-3′ (SEQ ID NO: 2174). Primers and probe for IL-12, designed using GenBank Accession number M86671.1, were: forward primer, 5′-GCCAGTACACCTGCCACAAA-3′ (SEQ ID NO: 2175), reverse primer, 5′-GACCAAATTCCATTTTCCTTCTTG-3′ (SEQ ID NO: 2176), probe 5′-FAM-AGGCGAGACTCTGAGCCACTCACATCTG-TAMRA-3′ (SEQ ID NO: 2177). Primers and probe for IL-6, designed using GenBank Accession number X54542.1, were: forward primer, 5′-CCTAGTGCGTTATGCCTAAGCA-3′ (SEQ ID NO: 2178), reverse primer, 5′-TTCGTAGAGAACAACATAAGTCAGATACC-3′ (SEQ ID NO: 2179), probe 5′-FAM-TTTCTGACCACAGTGAGGAATGTCCACAA-TAMRA-3′ (SEQ ID NO: 2180). The amount of total RNA in each sample was determined using a Ribogreen Assay (Molecular Probes, Eugene, Oreg.), and expression levels of TLR4, IL-12 and IL-6 were normalized to total RNA.

TLR4 is the primary macrophage receptor for LPS. Thus, ISIS Number 229927, targeted to TLR4, was tested for its ability to inhibit TLR4 expression and interfere with the response of macrophages to LPS, both with and without pretreatment with IFN-gamma. The treatment of primary murine macrophages with ISIS Number 229927 at reduced the expression of TLR4 in a dose-dependent manner, in both LPS-stimulated and LPS- and IFN-gamma-stimulated cells. As judged by the dose-dependent reduction in IL-12, the response of macrophages to LPS was reduced following inhibition of the TLR4 receptor expression, in both LPS-stimulated and LPS- and IFN-gamma-stimulated cells. These results demonstrated that ISIS 229927 can be used as a positive control for the inhibition of IL-12 expression in macrophages responding to LPS.

Primary mouse macrophages were treated with a selected group of oligomeric compounds targeting various miRNAs. These compounds and their miRNA targets are shown in Table 72. Table 72 shows IL-12 mRNA expression following treatment with 300 nM of oligomeric compounds and LPS (−IFN), and IL-12 mRNA expression following treatment with 300 nM of oligomeric compounds and stimulation with IFN-gamma and LPS (+IFN). The “−IFN” data represents a single experiment, and the “+IFN” data represents the average of 2 experiments. Data were normalized to values from cells receiving no oligomeric compound that were treated with LPS. IL-12 expression in cells receiving neither oligomeric compound nor LPS treatment was 2% of the control, both with and without IFN-gamma pretreatment, demonstrating that IL-12 mRNA expression was not stimulated in the absence of LPS treatment. Where present, “N.D.” indicates “not determined”.

TABLE 72 IL-12 mRNA expression in primary macrophages treated with oligomeric compounds targeting miRNAs and stimulated with LPS ISIS SEQ ID −IFN +IFN NO: NO: pri-miRNA % UTC % UTC 289606 863 Scrambled control N.D. 129 342673 758 mismatch to mir-143 91 N.D. 129690 907 Universal control 73 129 229927 2171 TLR4 92 145 327874 292 mir-30a 202 15 327876 294 mir-29b-1 194 9 327883 301 mir-27b 266 39 327887 305 mir-132 287 33 327889 307 mir-23b 153 10 327890 308 let-7i 183 94 327893 311 let-7b 117 52 327899 317 mir-183 164 7 327901 319 mir-143 225 9 327903 321 let-7a-3 200 23 327912 330 let-7f-1 206 39 327913 331 mir-29c 276 73 327917 335 mir-21 225 35 327919 337 mir-221 179 37 327920 338 mir-222 171 68 327921 339 mir-30d 325 24 327923 341 mir-128b 269 134 327924 342 mir-129-2 171 88 327925 343 mir-133b 302 60 327927 345 mir-15b 164 33 327928 346 mir-29a-1 201 61 327931 349 let-7c 105 48 327935 353 mir-20 254 24 327936 354 mir-133a-1 221 55 327940 358 mir-199a-2 228 107 327941 359 mir-181b 89 34 327945 363 mir-24-2 202 68 327956 374 mir-216 212 59 327958 376 mir-187 188 60 327959 377 mir-210 183 20 327961 379 mir-223 203 10 327963 381 mir-26b 224 23 327967 385 let-7g 203 43 327971 389 mir-23a 146 17 328105 407 hypothetical miRNA-088 108 57 328110 412 hypothetical miRNA-107 221 8 328117 419 hypothetical miRNA-144 162 72 328123 425 hypothetical miRNA-166 176 14 328129 431 hypothetical miRNA-173 87 10 328133 435 hypothetical miRNA-178 165 62 328137 439 hypothetical miRNA-183 213 12 328138 440 hypothetical miRNA-185 277 31 340341 236 mir-104 (Mourelatos) 139 13 340345 1882 miR-27 (Mourelatos) 104 78 341786 1845 miR-149 266 99 341790 1843 miR-154 318 84 341793 1836 miR-142-as 202 147 341800 1766 miR-186 180 100 341811 1906 miR-194 154 88 341815 1831 miR-200a 190 157

A comparison of the data from IFN-gamma-stimulated and unstimulated cells reveals that many of the oligomeric compounds targeting miRNAs attenuated the response of macrophages to LPS, as judged by IL-12 mRNA expression, when the cells were activated with IFN-gamma prior to LPS treatment. When macrophages were pretreated with IFN-gamma, treatment with several of the oligomeric compounds, such as ISIS Number 328110, ISIS Number 327901, ISIS Number 327899, ISIS Number 327876 and ISIS Number 327961 resulted in a reduction in IL-12 mRNA expression ranging from 20-fold to 30-fold. Other oligomeric compounds, such as ISIS Number 341800, ISIS Number 341811, ISIS Number 341793, ISIS Number 340345 and ISIS Number 341815 resulted in a less pronounced reduction in IL-12 mRNA expression ranging from 1.2-fold to 2-fold.

In a further embodiment, oligomeric compounds ISIS Number 327941 targeting mir-181b and ISIS Number 327921 targeting mir-30d were selected for a dose response study in LPS-stimulated primary macrophages, with and without IFN-gamma pre-treatment. Cells were treated as described herein, with oligomeric compound doses of 75, 150, 300 and 600 nM. Untreated control cells received no oligomeric compound treatment but did receive LPS treatment. ISIS 229927 (SEQ ID NO: 2171) was used as a positive control and ISIS 342683 (SEQ ID NO: 790), ISIS 126690 (SEQ ID NO: 907) and ISIS 289606 (SEQ ID NO: 863) were used as negative controls. IL-12 and IL-6 mRNA expression levels were measured by real-time PCR and normalized to untreated control cells that received LPS treatment. The IL-12 expression data, shown in Table 73, represent the average of 3 treatments. In cells receiving neither oligomeric compound nor LPS treatment, IL-12 expression was undetectable in IFN-gamma stimulated cells and was 1% of the untreated control in unstimulated cells.

TABLE 73 IL-12 mRNA expression following treatment of primary mouse macrophages with oligomeric compounds targeting mir-181b and mir-30d and LPS: dose response study IL-12 mRNA expression, % UTC SEQ Dose of oligomeric compound ISIS ID 75 nM 150 nM 300 nM 600 nM NO: NO: −IFN +IFN −IFN +IFN −IFN +IFN −IFN +IFN 327941 359 49 4 45 2 34 3 41 3 327921 339 109 14 88 7 67 5 53 5 229927 2171 67 46 53 35 45 16 46 8 342683 790 121 92 165 76 147 65 130 64 129690 907 114 66 109 54 101 66 128 81 289606 863 89 59 99 46 80 52 98 66

These data reveal that ISIS Number 327941 inhibited IL-12 expression in cells stimulated with LPS alone, where the percentage of untreated control ranged from 34% to 49%. ISIS Number 327921 inhibited IL-12 mRNA expression in a dose-dependent manner in cells stimulated with LPS alone, with the lowest IL-12 expression at 53% of untreated control. In cells pretreated with IFN-gamma and subsequently treated with LPS, ISIS Number 327941 markedly reduced IL-12 mRNA expression to less than 5% of the untreated control at all doses. ISIS Number 327921 reduced IL-12 expression to 14% of the control at all 75 nM and to less than 10% of the untreated control at all other doses. Thus, ISIS Number 327941, targeting mir-181b, and ISIS Number 327921, targeting mir-30d, resulted in a greater reduction in IL-12 expression than ISIS 229927, which is targeted to TLR4.

The IL-6 expression data, shown in Table 74, represents the average of 3 treatments. In cells receiving neither oligomeric compound nor LPS treatment, IL-12 expression was undetectable in IFN-gamma stimulated cells and was 2% of the untreated control in unstimulated cells.

TABLE 74 IL-6 mRNA expression following treatment of primary mouse macrophages with oligomeric compounds targeting mir-181b and mir-30d and LPS: dose response study IL-6 mRNA expression, % UTC SEQ Dose of oligomeric compound ISIS ID 75 nM 150 nM 300 nM 600 nM NO: NO: −IFN +IFN −IFN +IFN −IFN +IFN −IFN +IFN 327941 359 293 181 325 197 271 197 501 301 327921 339 223 122 294 144 522 287 632 313 229927 2171 57 54 52 39 44 40 104 69 342683 790 135 115 161 86 156 110 311 149 129690 907 98 92 99 86 109 94 258 203 289606 863 77 78 68 69 65 70 77 59

These data reveal that, in contrast to IL-12 expression, IL-6 expression is increased in a dose-dependent manner following treatment with ISIS Number 327941 and ISIS Number 327921, in both IFN-gamma-stimulated and unstimulated cells. This is in contrast to treatment with ISIS 229927, which exhibited some reduction in IL-6 expression in both IFN-gamma-stimulated and unstimulated cells.

Abnormalities in the signaling pathways controlling the expression of cytokines and cytokine receptors have been implicated in a number of diseases. Compounds that modulate the activity of macrophages, for example, the response to foreign antigens such as LPS, are candidate therapeutic agents with application in the treatment of conditions involving macrophage activation, such as septic shock and toxic shock

The expression of mir-181 in mouse cells and tissues was evaluated by Northern blot. Mouse tissues RNA was purchased from Ambion, Inc. (Austin, Tex.). RNA was prepared from macrophages were prepared and stimulated with LPS as described herein. Northern blotting was performed as described herein, and mir-181 levels were normalized to U6 levels, both of which were quantitated by Phosphorimager analysis. Expression levels are presented in arbitrary units. mir-181 was found to be most highly expressed in lung and kidney, at approximately equal levels. The next highest expression levels were found in brain, heart and liver. For example, as compared to kidney mir-181 expression levels, mir-181 was expressed approximately 2.5-fold lower in brain, approximately 2.2-fold lower in heart and approximately 1.8-fold lower in liver. mir-181 levels in both naïve and LPS-stimulated macrophages were 4.5-fold and 4.9-fold lower than in kidney, respectively. The lowest expression levels were found in thymus and spleen, which were 12.9-fold and 14.7-fold less as compared to kidney.

Example 40 Adipocyte Assay of Oligomeric Compounds

The effect of several oligomeric compounds of the present invention targeting miRNA target nucleic acids on the expression of markers of cellular differentiation was examined in differentiating adipocytes.

As described in Example 13, some genes known to be upregulated during adipocyte differentiation include HSL, aP2, Glut4 and PPARγ. These genes play important roles in the uptake of glucose and the metabolism and utilization of fats. An increase in triglyceride content is another well-established marker for adipocyte differentiation.

For assaying adipocyte differentiation, expression of the four hallmark genes, HSL, aP2, Glut4, and PPARγ, as well as triglyceride (TG) accumulation were measured as previously described in adipocytes transfected with oligomeric compounds targeting miRNAs. Triglyceride levels as well as mRNA levels for each of the four adipocyte differentiation hallmark genes are expressed as a percentage of untreated control (UTC) levels. In this experiment, the negative control oligomeric compound was ISIS Number 342672 (SEQ ID NO: 789) or ISIS Number 342673 (SEQ ID NO: 758). Results are shown in Table 75. Each value represents at least one oligomeric compound treatment; data from more than one oligomeric compound treatment were averaged. Where present, “N.D.” indicates “not determined”.

TABLE 75 Effects of oligomeric compounds targeting miRNAs on expression of adipocyte differentiation markers Isis PPAR Number SEQ ID NO Pri-miRNA TG HSL aP2 GLUT4 gamma UTC N/A N/A 100 100 100 100 100 327873 291 mir-140 105 116 113 106 104 327879 297 mir-7-1/mir-7-1* 59 103 103 99 81 327881 299 mir-128a 91 93 95 97 98 327885 303 mir-17/mir-91 29 57 69 40 59 327886 304 mir-123/mir-126 12 22 19 13 25 327887 305 mir-132 54 53 60 43 81 327891 309 mir-212 22 52 56 47 50 327895 313 mir-122a 76 88 90 76 86 327896 314 mir-22 22 37 43 35 52 327897 315 mir-92-1 28 39 62 32 66 327898 316 mir-142 102 92 96 82 101 327899 317 mir-183 25 27 47 14 62 327900 318 mir-214 26 21 32 12 55 327902 320 mir-192-1 55 56 58 15 56 327906 324 mir-103-1 25 37 46 14 50 327907 325 mir-26a-1 19 21 29 6 49 327910 328 mir-107 24 32 35 16 39 327911 329 mir-106 59 71 76 48 75 327912 330 let-7f-1 112 95 101 79 78 327916 334 mir-124a-2 56 64 67 51 71 327917 335 mir-21 26 26 32 15 54 327918 336 mir-144 65 85 91 66 74 327920 338 mir-222 20 14 22 0 34 327921 339 mir-30d 56 76 76 36 75 327923 341 mir-128b 88 64 65 54 77 327929 347 mir-199b 65 68 62 49 71 327935 353 mir-20 41 61 60 47 67 327936 354 mir-133a-1 23 40 40 6 47 327940 358 mir-199a-2 62 67 62 43 64 327943 361 mir-18 112 109 106 87 98 327944 362 mir-220 38 55 71 28 64 327945 363 mir-24-2 48 41 43 26 51 327946 364 mir-211 82 76 73 68 81 327949 367 mir-10a 43 49 52 20 54 327950 368 mir-19a 125 94 95 104 93 327952 370 mir-137 93 64 56 61 84 327957 375 mir-100-1 29 15 23 11 68 327958 376 mir-187 28 5 10 5 55 327959 377 mir-210 33 11 24 152 65 327961 379 mir-223 77 88 91 101 95 327962 380 mir-30c-1 64 77 75 58 80 327963 381 mir-26b 124 89 75 91 91 327964 382 mir-152 60 102 96 114 93 327965 383 mir-135-1 116 84 67 88 91 327966 384 mir-217 52 56 53 43 77 327968 386 sterol regulatory 94 79 67 85 79 element-binding protein-1/mir-33b 327969 387 mir-182 34 45 44 36 67 327970 388 mir-148a 48 25 29 27 46 327971 389 mir-23a 45 38 49 60 69 327972 390 mir-181c 67 70 70 75 85 328089 391 hypothetical miR- 67 55 50 59 79 13/miR-190 328090 392 hypothetical miRNA-023 128 81 68 86 95 328091 393 hypothetical miRNA-30 48 40 46 26 85 328092 394 glutamate receptor, 134 80 74 78 86 ionotrophic, AMPA 3/ hypothetical miRNA-033 328094 396 hypothetical miRNA-040 65 74 68 83 94 328095 397 hypothetical miRNA-041 110 83 70 98 92 328096 398 hypothetical miRNA-043 74 76 71 79 89 328097 399 hypothetical miRNA-044 65 54 48 62 63 328098 400 hypothetical miRNA-055 39 28 23 25 54 328099 401 hypothetical miRNA-058 57 74 80 61 72 328100 402 hypothetical miRNA-070 20 49 47 39 48 328101 403 LOC 114614 containing 67 78 83 57 70 miR-155/hypothetical miRNA-071 328102 404 hypothetical miRNA-075 70 99 96 58 94 328103 405 hypothetical miRNA-079 113 87 96 86 83 328104 406 hypothetical miRNA-083 64 81 94 83 73 328105 407 DiGeorge syndrome 82 95 102 75 85 critical region gene 8/hypothetical miRNA- 088 328106 408 hypothetical miRNA-090 70 86 91 79 81 328107 409 hypothetical miRNA-099 51 55 68 52 71 328108 410 hypothetical miRNA-101 79 75 87 65 72 328109 411 hypothetical miRNA-105 23 62 68 55 69 328110 412 hypothetical miRNA-107 96 84 89 77 80 328111 413 hypothetical miRNA-111 65 77 79 50 65 328113 415 hypothetical miRNA-137 74 83 87 78 85 328115 417 hypothetical miRNA-142 53 75 74 84 80 328116 418 hypothetical miRNA-143 107 91 99 105 95 328117 419 collagen, type I, 16 18 28 13 42 alpha 1/hypothetical miRNA-144 328118 420 hypothetical miRNA-153 69 67 74 57 72 328119 421 hypothetical miRNA-154 109 101 119 104 102 328120 422 hypothetical miRNA-156 80 67 80 68 73 328121 423 hypothetical miRNA-161 119 110 119 115 105 328122 424 hypothetical miRNA-164 97 89 99 91 103 328123 425 hypothetical miRNA-166 54 91 119 129 88 328124 426 hypothetical miRNA- 108 96 118 105 92 168-1/similar to ribosomal protein L5 328125 427 forkhead box 44 48 75 65 68 P2/hypothetical miRNA- 169 328126 428 hypothetical miRNA-170 108 135 120 107 98 328127 429 glutamate receptor, 81 93 95 75 85 ionotropic, AMPA 2/ hypothetical miRNA-171 328128 430 hypothetical miRNA-172 61 72 90 73 86 328129 431 hypothetical miRNA-173 19 34 54 36 59 328130 432 hypothetical miRNA-175 91 64 72 55 77 328131 433 hypothetical miRNA-176 74 51 63 56 55 328133 435 hypothetical miRNA-178 43 49 66 59 53 328134 436 hypothetical miRNA-179 107 109 97 109 86 328135 437 cezanne 2/ 29 20 34 19 33 hypothetical miRNA-180 328136 438 hypothetical miRNA-181 26 37 57 35 54 328137 439 tight junction protein 37 25 45 29 36 1 (zona occludens 1)/ hypothetical miRNA-183 328138 440 hypothetical miRNA-185 80 56 52 52 63 328139 441 hypothetical miRNA-188 90 116 100 85 91 340341 236 mir-104 (Mourelatos) 46 49 62 48 71 340343 1780 mir-105 (Mourelatos) 35 46 60 33 59 340348 848 mir-93 (Mourelatos) 48 57 68 52 78 340350 855 mir-95 (Mourelatos) 38 45 64 53 59 340352 1821 mir-99 (Mourelatos) 110 123 107 97 102 340354 1903 mir-25 64 56 72 61 74 340356 1853 mir-28 43 59 73 54 62 340358 1825 mir-31 23 24 47 21 42 340360 1865 mir-32 106 102 102 91 96 341791 1880 mir-30a 50 72 80 47 75 341795 1762 mir-199a-2 57 74 76 55 74 341796 1904 mir-131-1/mir-9 59 67 74 58 66 341797 1773 mir-17/mir-91 20 29 45 17 50 341798 1871 mir-123/mir-126 62 77 84 55 70 341799 1787 hypothetical miR- 98 103 101 89 89 13/miR-190 341800 1766 mir-186 18 42 50 28 61 341801 1839 mir-198 65 89 90 77 82 341802 1806 mir-191 155 121 98 85 127 341803 760 mir-206 N.D. 79 85 73 68 341804 761 mir-94/mir-106b N.D. 75 78 62 71 341805 762 mir-184 N.D. 86 90 74 77 341806 763 mir-195 N.D. 77 83 58 70 341807 764 mir-193 N.D. 102 82 101 83 344268 1774 mir-10b 57 44 46 22 53 344269 1890 mir-29c 42 35 41 28 48 344275 1912 mir-203 36 39 36 21 46 344276 1828 mir-204 66 68 72 49 72 344277 1767 mir-1d-2 75 57 61 45 68 344338 1812 mir-130a 103 89 86 66 91 344340 1921 mir-140 60 47 82 16 67 344341 1823 mir-218-1 50 33 42 14 49 344342 1814 mir-129-2 88 87 88 71 83 344343 1811 mir-130b 32 22 25 4 30 344611 1785 mir-240* (Kosik) 43 31 34 3 34 344612 1790 mir-232* (Kosik) 69 59 72 40 62 344613 1775 mir-227* (Kosik)/mir- 47 46 55 38 57 226* (Kosik) 344614 1834 mir-227* (Kosik)/mir- 89 71 78 61 86 226* (Kosik) 344615 1900 mir-244* (Kosik) 149 154 166 145 144 344616 1800 mir-224* (Kosik) 32 23 26 2 36 344617 1862 mir-248* (Kosik) 52 55 59 42 72 346685 1884 mir-27 (Mourelatos) 164 172 181 233 138 346686 1857 mir-101-1 73 80 83 73 83 346687 1802 mir-129-1 55 53 56 35 60 346688 1898 mir-182 33 39 48 12 55 346689 1830 mir-200b 59 63 79 45 64 346691 1870 mir-147 (Sanger) 56 69 69 64 79 346692 1889 mir-224 (Sanger) 35 18 26 11 28 346693 1838 mir-134 (Sanger) 69 66 77 65 81 346694 1763 mir-146 (Sanger) 31 18 41 5 32 346695 1824 mir-150 (Sanger) 69 73 72 58 78 346906 1781 mir-296 (RFAM/mmu) 83 70 77 70 80 346907 1815 mir-299 (RFAM/mmu) 47 36 50 37 51 346908 1881 mir-301 (RFAM/mmu) 75 71 77 65 77 346909 1902 mir-302 (RFAM/mmu) 66 64 68 64 77 346910 1866 mir-34a (RFAM/mmu) 80 69 78 63 83 346913 1795 let-7d 63 58 66 40 59 346914 1810 mir-94/mir-106b 41 27 48 16 41 346915 1784 mir-200a 73 67 83 75 90 346917 1826 mir-31 39 27 33 20 31 346919 1849 mir-93 (Mourelatos) 44 45 64 50 65 346920 1801 mir-96 63 53 70 61 70 346921 1759 mir-34 52 49 69 51 62 348116 1922 mir-320 43 58 79 48 76 348117 1860 mir-321-1 66 55 70 73 65 348119 1908 mir-142 91 76 81 86 90 348124 1820 mir-10b 53 43 59 41 63 348125 1878 mir-19b-1 79 64 67 65 64 348127 1869 mir-27b 155 150 185 201 130

Several compounds were found to have effects on adipocyte differentiation. For example, the oligomeric compounds ISIS Number 340348 (SEQ ID NO: 848), targeted to mir-93 (Mourelatos); ISIS Number 341798 (SEQ ID NO: 1871), targeted to mir-123/mir-126; ISIS Number 344340 (SEQ ID NO: 1921) targeted to mir-140; ISIS Number 346687 (SEQ ID NO: 1802), targeted to mir-129-1 and ISIS Number 348117 (SEQ ID NO: 1860), targeted to mir-321-1 were shown to significantly reduce the expression levels of 3 of the 5 markers of adipocyte differentiation. The effects of ISIS Number 327897 (SEQ ID NO: 315), targeted to mir-92-1, were even more pronounced, as shown by the significant reduction in expression of 4 of the 5 markers of differentiation. These data indicate that these oligomeric compounds have the ability to block adipocyte differentiation. Therefore, these oligomeric compounds may be useful as pharmaceutical agents with applications in the treatment, attenuation or prevention of obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases as well as having potential applications in the maintenance of the pluripotent phenotype of stem or precursor cells.

Other compounds were shown to stimulate adipocyte differentiation. For example, the oligomeric compounds ISIS Number 328121 (SEQ ID NO: 423), targeted to hypothetical miRNA-161; ISIS Number 344615 (SEQ ID NO: 1900), targeted to mir-244* (Kosik); ISIS Number 346685 (SEQ ID NO: 1884), targeted to mir-27 (Mourelatos); and ISIS Number 348127 (SEQ ID NO: 1869), targeted to mir-27b resulted in significant increases in all 5 markers of adipocyte differentiation. Other oligomeric compounds, for example ISIS Number 340352 (SEQ ID NO: 1821), targeted to mir-99 (Mourelatos) and ISIS Number 328126 (SEQ ID NO: 428), targeted to hypothetical miRNA-170, resulted in increases in 4 of the 5 markers of adipocyte differentiation. These oligomeric compounds may be useful as a pharmaceutical agents in the treatment of diseases in which the induction of adipocyte differentiation is desirable, such as anorexia, or for conditions or injuries in which the induction of cellular differentiation is desirable, such as Alzheimers disease or central nervous system injury, in which regeneration of neural tissue (such as from pluripotent stem cells) would be beneficial. Furthermore, this oligomeric compound may be useful in the treatment, attenuation or prevention of diseases in which it is desirable to induce cellular differentiation and/or quiescence, for example in the treatment of hyperproliferative disorders such as cancer.

In a further embodiment, oligomeric compounds of the present invention were tested for their effects on insulin signaling in HepG2 cells. As described in Example 18, insulin is known to regulate the expression of hepatic IGFBP-1, PEPCK-c and follistatin. Thus, the IGFBP-1, PEPCK-c and follistatin genes serve as marker genes for which mRNA expression can be monitored and used as an indicator of an insulin-resistant state. Oligomeric compounds with the ability to reduce expression of IGFBP-1, PEPCK-c and follistatin are highly desirable as agents potentially useful in the treatment of diabetes and hypertension oligomeric compounds of the invention were tested for their effects on insulin signalling in liver-derived cells. For assaying insulin signalling, expression of IGFBP-1, PEPCK-c and follistatin mRNAs were measured as previously described in HepG2 cells transfected with oligomeric compounds targeting miRNAs and treated with either no insulin (“basal” Experiment 1, for identification of insulin-mimetic compounds) or with 1 nM insulin (“insulin treated” Experiment 2, for identification of insulin sensitizers) for four hours. At the end of the insulin or no-insulin treatment, total RNA was isolated and real-time PCR was performed on all the total RNA samples using primer/probe sets for three insulin responsive genes: PEPCK-c, IGFBP-1 and follistatin. Expression levels for each gene are normalized to total RNA, and values are expressed relative to the transfectant only untreated control (UTC). In these experiments, the negative control oligomeric compound was ISIS Number 342672 (SEQ ID NO: 789) or ISIS Number 342673 (SEQ ID NO: 758). Results are shown in Tables 76 and 77. Each value represents at least one oligomeric compound treatment; data from more than one oligomeric compound treatment were averaged.

TABLE 76 Experiment 1: Effects of oligomeric compounds targeting miRNAs on insulin-repressed gene expression in HepG2 cells Isis SEQ ID Number NO Pri-miRNA Follistatin IGFBP1 PEPCKc UTC N/A N/A 100 100 100 327873 291 mir-140 97 108 72 327885 303 mir-17/mir-91 74 161 73 327886 304 mir-123/mir-126 82 176 61 327887 305 mir-132 113 119 83 327893 311 let-7b 93 107 81 327895 313 mir-122a 83 108 71 327897 315 mir-92-1 129 163 72 327899 317 mir-183 66 105 42 327900 318 mir-214 111 102 88 327911 329 mir-106 81 157 52 327916 334 mir-124a-2 108 102 88 327918 336 mir-144 75 95 81 327920 338 mir-222 99 165 52 327923 341 mir-128b 86 116 83 327946 364 mir-211 103 108 90 327949 367 mir-10a 112 112 81 327950 368 mir-19a 83 109 65 327952 370 mir-137 93 123 70 327957 375 mir-100-1 69 143 59 327958 376 mir-187 91 119 73 327959 377 mir-210 98 124 139 327961 379 mir-223 113 150 98 327963 381 mir-26b 101 108 92 327964 382 mir-152 97 100 74 327965 383 mir-135-1 95 106 63 341800 1766 mir-186 105 114 71 341801 1839 mir-198 85 99 73 341802 1806 mir-191 136 186 98 341803 760 mir-206 68 107 110 341804 761 mir-94/mir-106b 63 162 44 341805 762 mir-184 63 105 40 341806 763 mir-195 75 128 79 341807 764 mir-193 102 129 97 341808 1861 mir-185 96 113 64

Under “basal” conditions (without insulin), treatments of HepG2 cells with oligomeric compounds of the present invention resulting in decreased mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that the oligomeric compounds have an insulin mimetic effect. Treatments with oligomeric compounds of the present invention resulting in an increase in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds inhibit or counteract the normal insulin repression of mRNA expression of these genes.

From these data, it is evident that the oligomeric compounds, ISIS Number 327886 (SEQ ID NO: 304), targeting mir-123/mir-126; ISIS Number 327899 (SEQ ID NO: 317), targeting mir-183; ISIS Number 327911 (SEQ ID NO: 329), targeting mir-106; ISIS Number 327920 (SEQ ID NO: 338), targeting mir-222; ISIS Number 341804 (SEQ ID NO: 761), targeting mir-94/mir-106b; and ISIS Number 341805 (SEQ ID NO: 762), targeting mir-184, for example, resulted in 39%, 58%, 48%, 48%, 56% and 60% reductions, respectively, in PEPCK-c mRNA, a marker widely considered to be insulin-responsive. Thus, these oligomeric compounds may be useful as pharmaceutic agents comprising insulin mimetic properties in the treatment, amelioration, or prevention of diabetes or other metabolic diseases.

Conversely, the results observed with the oligomeric compounds targeting mir-92-1 (ISIS Number 327897, SEQ ID NO: 315), mir-10a (ISIS Number 327949, SEQ ID NO: 367), mir-223 (ISIS Number 327961, SEQ ID NO: 379) and mir-191 (ISIS Number 341802, SEQ ID NO: 1806), for example, exhibited increased expression of the IGFBP-1 and follistatin marker genes, suggesting that the mir-92-1, mir-10a, mir-223, and mir-191 miRNA targets may be involved in the regulation of these insulin-responsive genes. When these miRNAs are inactivated by an oligomeric compound, IGFBP-1 and follistatin gene expression is no longer repressed. Similarly, treatment oligomeric compounds targeting mir-210 (ISIS Number 327959, SEQ ID NO: 377)) and mir-206 (ISIS Number 341803, SEQ ID NO: 760) resulted in increases in the IGFBP-1 and PEPCK-c marker genes, suggesting that mir-210 and mir-206 may be involved in the regulation of these insulin-responsive genes.

TABLE 77 Experiment 2: Effects of oligomeric compounds targeting miRNAs on insulin-sensitization of gene expression in HepG2 cells Isis SEQ ID Number NO Pri-miRNA Follistatin IGFBP1 PEPCKc UTC + N/A N/A 100 100 100 1 nM insulin 327897 315 mir-92-1 123 243 78 327911 329 mir-106 71 160 78 327916 334 mir-124a-2 98 128 88 327918 336 mir-144 76 81 107 327920 338 mir-222 102 267 59 327923 341 mir-128b 106 119 125 327946 364 mir-211 109 138 99 327949 367 mir-10a 111 172 101 327950 368 mir-19a 89 124 82 327952 370 mir-137 100 103 85 327957 375 mir-100-1 73 184 88 327958 376 mir-187 112 149 106 327959 377 mir-210 92 141 156 327961 379 mir-223 128 160 126 327963 381 mir-26b 95 111 94 327964 382 mir-152 114 121 122 327965 383 mir-135-1 79 105 64 328114 416 hypothetical 81 177 41 miRNA-138 328115 417 hypothetical 91 120 59 miRNA-142 328125 427 forkhead box 107 216 77 P2/hypothetical miRNA-169 328342 451 mir-203 88 98 39 328343 452 mir-7-1/mir-7-1* 139 135 69 328358 467 mir-123/mir-126 106 165 93 328367 476 mir-212 107 141 85 328377 486 hypothetical 159 247 182 miRNA-30 328396 505 mir-205 135 128 65 328397 506 mir-103-1 75 57 76 328423 532 mir-19b-2 114 69 77 328649 558 mir-20 69 115 86 328702 611 mir-10a 88 83 96 328761 670 hypothetical 53 193 64 miRNA-138 328764 673 hypothetical 128 145 68 miRNA-142 328769 678 mir-26b 84 110 100 328774 683 sterol regulatory 68 100 77 element-binding protein-1/mir-33b 328776 685 forkhead box 114 86 125 P2/hypothetical 1 miRNA-69

For HepG2 cells treated with 1 nM insulin, treatments with oligomeric compounds of the present invention resulting in a decrease in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds have an insulin sensitization effect. Treatments with oligomeric compounds of the present invention resulting in an increase in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds inhibit or counteract the normal insulin response of repression of mRNA expression of these genes.

From these data, it is evident that the oligomeric compounds, ISIS Number 327920 (SEQ ID NO: 338), targeting mir-222; ISIS Number 328114 (SEQ ID NO: 416), targeting hypothetical miRNA-138; ISIS Number 328115 (SEQ ID NO: 417), targeting hypothetical miRNA-142; and ISIS Number 328342 (SEQ ID NO: 451) targeting mir-203, for example, were observed to result in a 41%, a 59%, a 41% and a 61% reduction, respectively, of PEPCK-c mRNA expression, widely considered to be a marker of insulin-responsiveness. Thus, these oligomeric compounds may be useful as pharmaceutic agents with insulin-sensitizing properties in the treatment, amelioration, or prevention of diabetes or other metabolic diseases.

Conversely, the results observed with the oligomeric compounds targeting mir-128b (ISIS Number 327923, SEQ ID NO: 341), mir-223 (ISIS Number 327961, SEQ ID NO: 379), mir-152 (ISIS Number 327964, SEQ ID NO: 382) and hypothetical miRNA-30 (ISIS Number 328377, SEQ ID NO: 486), all exhibiting increased expression of the IGFBP-1, PEPCK-c and follistatin marker genes, support the conclusion that the mir-128b, mir-223, mir-152 and hypothetical miRNA-30 may be involved in the regulation of insulin-responsive genes. When these miRNAs are inactivated by the oligomeric compounds of the present invention, IGFBP-1, PEPCK-c and follistatin gene expression is no longer repressed or insulin-sensitive.

Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank accession numbers, and the like) cited in the present application is incorporated herein by reference in its entirety. 

1. A method of inhibiting the activity of a microRNA comprising contacting a cell comprising the microRNA with a compound comprising an oligomeric compound, wherein: the oligomeric compound is at least 90% complementary to a miRNA or a precursor thereof; the oligomeric compound consists of 15 to 30 linked monomeric subunits; at least one monomeric subunit is a modified nucleoside; and the miRNA is selected from miR-21 (SEQ ID NO: 64) and miR-1 (SEQ ID NO: 29).
 2. The method of claim 1, wherein the miRNA is miR-21 (SEQ ID NO: 64).
 3. The method of claim 1, wherein the miRNA is miR-1 (SEQ ID NO: 29).
 4. The method of claim 1 wherein the oligomeric compound is at least 95% complementary to the miRNA.
 5. The method of claim 1 wherein the oligomeric compound is 100% complementary to the miRNA.
 6. The method of claim 1 wherein the oligomeric compound is 100% complementary to at least an 8-nucleobase portion of the miRNA.
 7. The method of claim 1 wherein the oligomeric compound consists of 19 to 23 linked monomeric subunits.
 8. The method of claim 1 wherein the oligomeric compound consists of an oligonucleotide.
 9. The method of claim 8 wherein the oligonucleotide consists of 19 to 23 linked nucleosides.
 10. The method of claim 1 wherein the oligomeric compound comprises at least one modified sugar moiety.
 11. The method of claim 10 wherein the modified sugar moiety is selected from 2′-F, 2′-O-methyl, 2′-O-methoxyethyl, and a bicyclic sugar moiety.
 12. The method of claim 11 wherein the bicyclic sugar moiety comprises a 4′-CH₂—O-2′ bridge.
 13. The method of claim 1 wherein the oligomeric compound comprises at least one modified internucleoside linkage.
 14. The method of claim 13 wherein the modified internucleoside linkage is a phosphorothioate linkage.
 15. The method of claim 1 wherein each internucleoside linkage of the oligomeric compound is a phosphorothioate linkage.
 16. The method of claim 1 wherein each monomeric subunit comprises a modified sugar moiety.
 17. The method of claim 1 wherein the oligonucleotide comprises two or more chemically distinct regions.
 18. The method of claim 17 wherein each nucleoside of the two or more chemically distinct regions is independently selected from a 2′-fluoro nucleoside, a 2′-O-methyl nucleoside, a 2′-O-methoxyethyl nucleoside, a 2′-deoxynucleoside, and a bicyclic sugar nucleoside.
 19. The method of claim 17 wherein each nucleoside of the two or more chemically distinct regions is independently selected from a 2′-deoxynucleoside and a bicyclic sugar nucleoside.
 20. The method of claim 19 wherein the bicyclic nucleoside comprises a 4′-CH₂—O-2′ bridge.
 21. The method of claim 1 wherein the oligomeric compound comprises at least one modified nucleobase.
 22. The method of claim 21 wherein the modified nucleobase is 5-methylcytosine.
 23. The method of claim 1 wherein the oligomeric compound is attached to a conjugate group.
 24. The method of claim 23 wherein the conjugate group is cholesterol.
 25. A composition comprising a compound comprising an oligomeric compound, wherein: the oligomeric compound is at least 90% complementary to a miRNA or a precursor thereof; the oligomeric compound consists of 15 to 30 linked monomeric subunits; at least one monomeric subunit is a modified nucleoside; and the miRNA is selected from miR-21 (SEQ ID NO: 64) and miR-1 (SEQ ID NO: 29); and a pharmaceutically acceptable diluent or carrier. 